Crystalline forms

ABSTRACT

Provided herein are compound of Formula I-IV and pharmaceutically acceptable salts thereof which exhibit rearranged during transfection (RET) kinase inhibition. In particular, provided herein are novel crystalline forms of 4-(6-(4-((6-methoxypyridin-3-yl)methyl)piperazin-1-yl)pyridin-3-yl)-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile (Formula I), 6-(2-hydroxy-2-methylpropoxy)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile (Formula II), 6-(2-hydroxy-2-methylpropoxy)-4-(6-(6-(6-methoxynicotinoyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile (Formula III), 6-(2-hydroxy-2-methylpropoxy)-4-(6-(4-hydroxy-4-(pyridin-2-ylmethyl)piperidin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile (Formula IV), and pharmaceutically acceptable salts thereof, pharmaceutical compositions comprising the compounds, processes for making the compounds, and the use of the compounds in therapy. More particularly, the application relates to novel crystalline forms of Formula I-IV and pharmaceutically acceptable salts thereof useful in the treatment and prevention of diseases which can be treated with a RET kinase inhibitor, including RET-associated diseases and disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 62/570,573, filed on Oct. 10, 2017; 62/643,950, filed on Mar. 16,2018; 62/656,668, filed on Apr. 12, 2018; 62/669,288, filed on May 9,2018; 62/676,417, filed on May 25, 2018; and 62/712,707, filed on Jul.31, 2018; the contents of each of which are hereby incorporated byreference in their entireties.

BACKGROUND

RET is a single-pass transmembrane receptor belonging to the tyrosinekinase superfamily that is required for normal development, maturation,and maintenance of several tissues and cell types (Mulligan, L. M.,Nature Reviews Cancer (2014) 14:173-186). The extracellular portion ofthe RET kinase contains four calcium-dependent cadherin-like repeatsinvolved in ligand binding and a juxtamembrane cysteine-rich regionnecessary for the correct folding of the RET extracellular domain, whilethe cytoplasmic portion of the receptor includes two tyrosine kinasesubdomains.

RET signaling is mediated by the binding of a group of soluble proteinsof the glial cell line-derived neurotrophic factor (GDNF) family ligands(GFLs), which also includes neurturin (NTRN), artemin (ARTN), andpersephin (PSPN) (Arighi et al., Cytokine Growth Factor Rev. (2005)16:441-67). Unlike other receptor tyrosine kinases, RET does notdirectly bind to GFLs and requires an additional co-receptor: that is,one of four GDNF family receptor-α (GFRα) family members, which aretethered to the cell surface by a glycosylphosphatidylinositol linkage.GFLs and GFRα family members form binary complexes that in turn bind toRET and recruit it into cholesterol-rich membrane subdomains, which areknown as lipid rafts, where RET signaling occurs.

Upon binding of the ligand-co-receptor complex, RET dimerization andautophosphorylation on intracellular tyrosine residues recruits adaptorand signaling proteins to stimulate multiple downstream pathways.Adaptor protein binding to these docking sites leads to activation ofRas-MAPK and PI3K-Akt/mTOR signaling pathways or to recruitment of theCBL family of ubiquitin ligases that functions in RET downregulation ofthe RET-mediated functions.

Aberrant RET expression and/or activity have been demonstrated indifferent cancers and in gastrointestinal disorders such as irritablebowel syndrome (IBS).

SUMMARY

Compounds of Formula I-IV,4-(6-(4-((6-methoxypyridin-3-yl)methyl)piperazin-1-yl)pyridin-3-yl)-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile(Formula I);6-(2-hydroxy-2-methylpropoxy)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile(Formula II);6-(2-hydroxy-2-methylpropoxy)-4-(6-(6-(6-methoxynicotinoyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile(Formula III); and6-(2-hydroxy-2-methylpropoxy)-4-(6-(4-hydroxy-4-(pyridin-2-ylmethyl)piperidin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile(Formula IV) are inhibitors of RET kinase, and are useful for treatingdiseases such as proliferative diseases, including cancers.

Accordingly, provided herein is a compound of Formula I-IV:

and pharmaceutically acceptable salts, amorphous, and polymorph formsthereof.

Also provided herein is a crystalline form of a compound of Formula I,wherein the crystalline form is Form A, and is characterized by havingan X-ray powder diffraction (XRPD) pattern comprising peaks at °2θvalues of 4.4±0.2, 14.6±0.2, and 18.3±0.2.

Also provided herein is a crystalline form of a compound of Formula II,wherein the crystalline form is Form 1, and is characterized by havingan X-ray powder diffraction (XRPD) pattern comprising peaks at °2θvalues of 16.5±0.2, 18.9±0.2, and 26.0±0.2.

Also provided herein is a crystalline form of a compound of Formula III,wherein the crystalline form is Form A, and is characterized by havingan X-ray powder diffraction (XRPD) pattern comprising peaks at °2θvalues of 17.3±0.2, 19.2±0.2, and 23.9±0.2.

Also provided herein is a crystalline form of a compound of Formula IV,wherein the crystalline form is Form A, and is characterized by havingan X-ray powder diffraction (XRPD) pattern comprising peaks at °2θvalues of 8.3±0.2, 16.3±0.2, and 21.9±0.2.

Also provided herein is a solid oral pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a compound ofFormula I-IV, including pharmaceutically acceptable salts, amorphous,and polymorph forms thereof.

Also provided herein is a liquid pharmaceutical composition comprising apharmaceutically acceptable carrier and a compound of Formula I-IV,including pharmaceutically acceptable salts, amorphous, and polymorphforms thereof.

Also provided herein is a method for treating cancer in a subject inneed thereof, the method comprising administering a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundof Formula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof, or a pharmaceutical composition prepared using acompound of Formula I-IV or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof.

Also provided herein is a method of inhibiting cell proliferation, invitro or in vivo, the method comprising contacting a cell with aneffective amount of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof, or apharmaceutical composition prepared using a compound of Formula I-IV ora pharmaceutically acceptable salt, amorphous, or polymorph formthereof, or a pharmaceutical composition thereof as defined herein.

Also provided herein is a method of treating a RET-associated disease ordisorder in a patient in need of such treatment, the method comprisingadministering to the patient a therapeutically effective amount of acompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, or a pharmaceutical compositionthereof as defined herein.

Also provided herein is a method of treating cancer and/or inhibitingmetastasis associated with a particular cancer in a patient in need ofsuch treatment, the method comprising administering to the patient atherapeutically effective amount of a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,or a pharmaceutical composition prepared using a compound of FormulaI-IV or a pharmaceutically acceptable salt, amorphous, or polymorph formthereof or a pharmaceutical composition thereof as defined herein.

Also provided herein is a method for treating cancer and/or inhibitingmetastasis associated with a particular cancer in a subject in needthereof, the method comprising (a) determining if the cancer isassociated with a dysregulation of a RET gene, a RET kinase, orexpression or activity or level of any of the same; and (b) if thecancer is determined to be associated with a dysregulation of a RETgene, a RET kinase, or expression or activity or level of any of thesame, administering to the subject a therapeutically effective amount ofa compound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, or a pharmaceutical compositioncomprising a compound of Formula I-IV, for example, a pharmaceuticalcomposition prepared using a compound of Formula I-IV or apharmaceutically acceptable salt, amorphous, or polymorph form thereof.

Also provided herein is a method of treating a RET-associated cancer ina subject, the method comprising administering to a subject identifiedor diagnosed as having a RET-associated cancer a therapeuticallyeffective amount of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof, or apharmaceutical composition comprising a compound of Formula I-IV, forexample, a pharmaceutical composition prepared using a compound ofFormula I-IV or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof.

Also provided herein is a method of treating a RET-associated cancer ina subject, the method comprising: determining if the cancer in thesubject is a RET-associated cancer; and administering to a subjectdetermined to have a RET-associated cancer a therapeutically effectiveamount of a compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof, or a pharmaceuticalcomposition comprising a compound of Formula I-IV, for example, apharmaceutical composition prepared using a compound of Formula I-IV ora pharmaceutically acceptable salt, amorphous, or polymorph formthereof.

Also provided herein is a method of treating a subject, the methodcomprising administering a therapeutically effective amount of acompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, or a pharmaceutical compositioncomprising a compound of Formula I-IV, for example, a pharmaceuticalcomposition prepared using a compound of Formula I-IV or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,to a subject having a clinical record that indicates that the subjecthas dysregulation of a RET gene, a RET kinase, or expression or activityor level of any of the same.

Also provided herein is a method of selecting a treatment for a subject,the method comprising selecting a treatment comprising administration ofa therapeutically effective amount of a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,or a pharmaceutical composition comprising a compound of Formula I-IV,for example, a pharmaceutical composition prepared using a compound ofFormula I-IV or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof, for a subject identified or diagnosed as havinga RET-associated cancer.

Also provided herein is a method of selecting a treatment for a subjecthaving a cancer, the method comprising: determining if the cancer in thesubject is a RET-associated cancer; and selecting a treatment includingadministration of a therapeutically effective amount of a compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof, or a pharmaceutical composition comprising acompound of Formula I-IV, for example, a pharmaceutical compositionprepared using a compound of Formula I-IV or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof, for a subjectdetermined to have a RET-associated cancer.

Also provided herein is a method of selecting a subject for treatmentincluding administration of a therapeutically effective amount of acompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, or a pharmaceutical compositioncomprising a compound of Formula I-IV, for example, a pharmaceuticalcomposition prepared using a compound of Formula I-IV or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,the method comprising: identifying a subject having a RET-associatedcancer; and selecting the subject for treatment including administrationof a therapeutically effective amount of a compound of Formula I-IV, ora pharmaceutically acceptable salt, amorphous, or polymorph formthereof, or a pharmaceutical composition comprising a compound ofFormula I-IV, for example, a pharmaceutical composition prepared using acompound of Formula I-IV or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof.

Also provided herein is a method of selecting a subject having cancerfor treatment including administration of a therapeutically effectiveamount of a compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof, or a pharmaceuticalcomposition comprising a compound of Formula I-IV, for example, apharmaceutical composition prepared using a compound of Formula I-IV ora pharmaceutically acceptable salt, amorphous, or polymorph formthereof, the method comprising: determining if the cancer in the subjectis a RET-associated cancer; and selecting a subject determined to have aRET-associated cancer for treatment including administration of atherapeutically effective amount of a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,or a pharmaceutical composition comprising a compound of Formula I-IV,for example, a pharmaceutical composition prepared using a compound ofFormula I-IV or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof.

Also provided herein is the use of a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,for the manufacture of a medicament for treating a RET-associated cancerin a subject.

Also provided herein is a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,or a pharmaceutical composition comprising a compound of Formula I-IV,for example, a pharmaceutical composition prepared using a compound ofFormula I-IV or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof, for use in treating a subject identified ordiagnosed as having a RET-associated cancer.

Also provided herein is a method for inhibiting RET kinase activity in amammalian cell, the method comprising contacting the mammalian cell witha compound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof.

Also provided herein is a method of treating irritable bowel syndrome ina subject, the method comprising administering to a subject identifiedor diagnosed as having irritable bowel syndrome a therapeuticallyeffective amount of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof, or apharmaceutical composition comprising a compound of Formula I-IV, forexample, a pharmaceutical composition prepared using a compound ofFormula I-IV or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof, to the subject.

Also provided herein is a method for reducing pain associated withirritable bowel syndrome in a subject in need thereof, the methodcomprising administering to a subject identified or diagnosed as havingirritable bowel syndrome a therapeutically effective amount of acompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, or a pharmaceutical compositioncomprising a compound of Formula I-IV, for example, a pharmaceuticalcomposition prepared using a compound of Formula I-IV or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,to the subject.

Also provided is a method of providing supportive care to a cancerpatient, including preventing or minimizing gastrointestinal disorders,such as diarrhea, associated with treatment, including chemotherapeutictreatment, the method comprising administering to the patient atherapeutically effective amount of a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,or a pharmaceutical composition prepared using a compound of FormulaI-IV or a pharmaceutically acceptable salt, amorphous, or polymorph formthereof or a pharmaceutical composition thereof as defined herein.

Also provided herein is a method for inhibiting metastasis (e.g., brainmetastasis) of a cancer in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of a compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof, or a pharmaceuticalcomposition comprising a compound of Formula I-IV, for example, apharmaceutical composition prepared using a compound of Formula I-IV ora pharmaceutically acceptable salt, amorphous, or polymorph formthereof. In some embodiments, the compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,or a pharmaceutical composition comprising a compound of Formula I-IV,for example, a pharmaceutical composition prepared using a compound ofFormula I-IV or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof, is used in combination with anotherchemotherapeutic agent.

Also provided herein is a method of treating a subject having a cancer,wherein the method comprises: (a) administering one or more doses of afirst RET inhibitor to the subject for a period of time; (b) after (a),determining whether a cancer cell in a sample obtained from the subjecthas at least one RET inhibitor resistance mutation that confersincreased resistance to a cancer cell or tumor to treatment with thefirst RET inhibitor of step (a); and (c) administering a compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof or a pharmaceutical composition comprising acompound of Formula I-IV, for example, a pharmaceutical compositionprepared using a compound of Formula I-IV or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof, as a monotherapyor in conjunction with another anticancer agent to the subject if thesubject has a cancer cell that has at least one RET inhibitor resistancemutation that confers increased resistance to a cancer cell or tumor totreatment with the first RET inhibitor of step (a); or (d) administeringadditional doses of the first RET inhibitor of step (a) to the subjectif the subject has a cancer cell that does not have a RET inhibitorresistance mutation that confers increased resistance to a cancer cellor tumor to treatment with the first RET inhibitor of step (a).

Also provided herein is a method of treating a subject having a cancer,wherein the method comprises: (a) administering one or more doses of afirst RET inhibitor, to the subject for a period of time; (b) after (a),determining whether a cancer cell in a sample obtained from the subjecthas at least one RET inhibitor resistance mutation that confersincreased resistance to a cancer cell or tumor to treatment with thefirst RET inhibitor of step (a); (c) administering a second RETinhibitor as a monotherapy or in conjunction with another anticanceragent to the subject if the subject has a cancer cell that has at leastone RET inhibitor resistance mutation that confers increased resistanceto a cancer cell or tumor to treatment with the first RET inhibitor ofstep (a); or (d) administering additional doses of the first RETinhibitor of step (a) to the subject if the subject has a cancer cellthat does not have a RET inhibitor resistance mutation that confersincreased resistance to a cancer cell or tumor to treatment with thefirst RET inhibitor of step (a); wherein the mutation is a substitutionat amino acid position 804, e.g., V804M, V804L, or V804E.

Also provided herein is a method of treating a subject having a cancer,wherein the method comprises: (a) determining whether a cancer cell in asample obtained from a subject having a cancer and previouslyadministered one or more doses of a first RET inhibitor has one or moreRET inhibitor resistance mutations that confer increased resistance to acancer cell or tumor to treatment with the first RET inhibitor that waspreviously administered to the subject; and (b) administering a compoundof Formula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof or a pharmaceutical composition comprising acompound of Formula I-IV, for example, a pharmaceutical compositionprepared using a compound of Formula I-IV or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof, as a monotherapyor in conjunction with another anticancer agent to the subject if thesubject has a cancer cell that has at least one RET inhibitor resistancemutation that confers increased resistance to a cancer cell or tumor totreatment with the first RET inhibitor that was previously administeredto the subject; or (c) administering additional doses of the first RETinhibitor to the subject if the subject has cancer cell that does nothave a RET inhibitor resistance mutation that confers increasedresistance to a cancer cell or tumor to treatment with the first RETinhibitor previously administered to the subject.

Also provided herein is a method of treating a subject having a cancer,wherein the method comprises: (a) determining whether a cancer cell in asample obtained from a subject having a cancer and previouslyadministered one or more doses of a first RET inhibitor has one or moreRET inhibitor resistance mutations that confer increased resistance to acancer cell or tumor to treatment with the first RET inhibitorpreviously administered to the subject; and (b) administering a secondRET inhibitor to the subject as a monotherapy or in conjunction withanother anticancer agent to the subject if the subject has a cancer cellthat has at least one RET inhibitor resistance mutation that confersincreased resistance to a cancer cell or tumor to treatment with thefirst RET inhibitor that was previously administered to the subject; or(c) administering additional doses of the first RET inhibitor that waspreviously administered to the subject if the subject has cancer cellthat does not have a RET inhibitor resistance mutation that confersincreased resistance to a cancer cell or tumor to treatment with thefirst RET inhibitor that was previously administered to the subject.

Also provided herein is a method of treating a subject having a cancer,wherein the method comprises: (a) administering one or more doses of acompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, or a pharmaceutical compositioncomprising a compound of Formula I-IV, for example, a pharmaceuticalcomposition prepared using a compound of Formula I-IV or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,for a period of time; (b) after (a), determining whether a cancer cellin a sample obtained from the subject has one or more RET inhibitorresistance mutations that confer increased resistance to a cancer cellor tumor to treatment with the compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,or the pharmaceutical composition comprising a compound of Formula I-IV,for example, a pharmaceutical composition prepared using a compound ofFormula I-IV or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof, of step (a); and (c) administering a second RETinhibitor or a second compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof, or apharmaceutical composition comprising a compound of Formula I-IV, forexample, a pharmaceutical composition prepared using a compound ofFormula I-IV or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof, as a monotherapy or in conjunction with anotheranticancer agent to a subject having a cancer cell that has one or moreRET inhibitor resistance mutations that confer increased resistance to acancer cell or tumor to treatment with the compound of Formula I-IV, ora pharmaceutically acceptable salt, amorphous, or polymorph formthereof, or the pharmaceutical composition comprising a compound ofFormula I-IV, for example, a pharmaceutical composition prepared using acompound of Formula I-IV or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, of step (a); or (d) administeringadditional doses of the compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof, or thepharmaceutical composition comprising a compound of Formula I-IV, forexample, a pharmaceutical composition prepared using a compound ofFormula I-IV or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof, of step (a) to a subject having a cancer cellthat does not have a RET inhibitor resistance mutation that confersincreased resistance to a cancer cell or tumor to treatment with thecompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, or the pharmaceutical compositioncomprising a compound of Formula I-IV, for example, a pharmaceuticalcomposition prepared using a compound of Formula I-IV or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,of step (a).

Also provided herein is a method of treating a subject having a cancer,wherein the method comprises: (a) determining whether a cancer cell in asample obtained from a subject having a cancer and previouslyadministered one or more doses of a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,has one or more RET inhibitor resistance mutations that confer increasedresistance to a cancer cell or tumor to treatment with the compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof, that was previously administered to the subject;(b) administering a second RET inhibitor or a second compound of FormulaI-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphform thereof, as a monotherapy or in conjunction with another anticanceragent to a subject having a cancer cell that has one or more RETinhibitor resistance mutations that confer increased resistance to acancer cell or tumor to treatment the compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,that was previously administered to the subject; or (c) administeringadditional doses of the compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof, previouslyadministered to a subject having a cancer cell that does not have a RETinhibitor resistance mutation that confers increased resistance to acancer cell or tumor to treatment with the compound of Formula I-IV, ora pharmaceutically acceptable salt, amorphous, or polymorph formthereof, that was previously administered to the subject.

Also provided herein is a pharmaceutical combination for treating cancer(e.g., a RET-associated cancer, such as a RET-associated cancer havingone or more RET inhibitor resistance mutations) in a patient in needthereof, which comprises (a) a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,or a pharmaceutical composition prepared using a compound of FormulaI-IV or a pharmaceutically acceptable salt, amorphous, or polymorph formthereof, (b) an additional therapeutic agent, and (c) optionally atleast one pharmaceutically acceptable carrier, wherein the compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof and the additional therapeutic are formulated asseparate compositions or dosages for simultaneous, separate orsequential use for the treatment of cancer, wherein the amounts of thecompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, or a pharmaceutical compositionprepared using a compound of Formula I-IV or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof and of theadditional therapeutic agent are together effective in treating thecancer. Also provided herein is a pharmaceutical composition comprisingsuch a combination. Also provided herein is the use of such acombination for the preparation of a medicament for the treatment ofcancer. Also provided herein is a commercial package or productcomprising such a combination as a combined preparation forsimultaneous, separate or sequential use; and to a method of treatmentof cancer a patient in need thereof.

Also provided herein is a method for reversing or preventing acquiredresistance to an anticancer drug, comprising administering atherapeutically effective amount of a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,or a pharmaceutical composition prepared using a compound of FormulaI-IV or a pharmaceutically acceptable salt, amorphous, or polymorph formthereof, to a patient at risk for developing or having acquiredresistance to an anticancer drug. In some embodiments, the patient isadministered a dose of the anticancer drug (e.g., at substantially thesame time as a dose of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof, or apharmaceutical composition prepared using a compound of Formula I-IV ora pharmaceutically acceptable salt, amorphous, or polymorph form thereofis administered to the patient).

Also provided herein is a method of delaying and/or preventingdevelopment of cancer resistant to an anticancer drug in an individual,comprising administering to the individual an effective amount of acompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, or a pharmaceutical compositionprepared using a compound of Formula I-IV or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof, before, during,or after administration of an effective amount of the anticancer drug.

Also provided herein is a method of treating an individual with cancerwho has an increased likelihood of developing resistance to ananticancer drug, comprising administering to the individual (a) aneffective amount of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof, before, during,or after administration of (b) an effective amount of the anticancerdrug.

Also provided are methods of treating an individual with aRET-associated cancer that has one or more RET inhibitor resistancemutations that increase resistance of the cancer to a first RETinhibitor (e.g., one or more amino acid substitutions in the kinasedomain (e.g., amino acid positions 700 to 1012 in a wildtype RETprotein), a gatekeeper amino acid (e.g., amino acid position 804 in awildtype RET protein), the P-loop (e.g., amino acid positions 730-737 ina wildtype RET protein), the X-DFG residue (e.g., amino acid position891 in a wildtype RET protein), ATP cleft solvent front amino acids(e.g., amino acid positions 806-811 in a wildtype RET protein), theactivation loop (e.g., amino acid positions 891-916 in a wildtype RETprotein), the C-helix and loop preceeding the C-helix (e.g., amino acidpositions 768-788 in a wildtype RET protein), and/or the ATP bindingsite (e.g., amino acid positions 730-733, 738, 756, 758, 804, 805, 807,811, 881, and 892 in a wildtype RET protein) (e.g., a substitution atamino acid position 804, e.g., V804M, V804L, or V804E, or a substitutionat amino acid position 810, e.g., G810S, G810R, G810C, G810A, G810V, andG810D, and/or one or more RET inhibitor resistance mutations listed inTables 3 and 4), that include administering a compound of Formula I-IV,or a pharmaceutically acceptable salt, amorphous, or polymorph formthereof, or a pharmaceutical composition prepared using a compound ofFormula I-IV or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof, before, during, or after administration ofanother anticancer drug (e.g., a second RET kinase inhibitor). See alsoJ. Kooistra, G. K. Kanev, O. P. J. Van Linden, R. Leurs, I. J. P. DeEsch, and C. De Graaf, “KLIFS: A structural kinase-ligand interactiondatabase,” Nucleic Acids Res., vol. 44, no. D1, pp. D365-D371, 2016; andO. P. J. Van Linden, A. J. Kooistra, R. Leurs, I. J. P. De Esch, and C.De Graaf, “KLIFS: A knowledge-based structural database to navigatekinase-ligand interaction space,” J. Med. Chem., vol. 57, no. 2, pp.249-277, 2014, both of which are incorporated by reference in theirentirey herein. In some embodiments, a wildtype RET protein is theexemplary wildtype RET protein described herein.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A-1E are plots of solubility of the compound of Formula I(freebase) in different solvent systems. FIG. 1A is a plot of solubilityin DCM/EtOH. FIG. 1B is a plot of solubility in DMSO/EtOH. FIG. 1C is aplot of solubility in DMSO/H₂O. FIG. 1D is a plot of solubility inTHF/EtOH. FIG. 1E is a plot of solubility in THF/H₂O.

FIGS. 2A-2G are scans of the freebase of the compound of Formula I. FIG.2A is an X-ray powder diffraction scan of the Form A of the compound ofFormula I (freebase). FIG. 2B is an overlay of X-ray powder diffractionscans of the freebase from different lots before and after DVS analysis.FIG. 2C is a differential calorimetry scan of the freebase. FIG. 2D isan isothermic (25° C.) dynamic vapor sorption scan of the freebase. FIG.2E is a thermogravimetric analysis scan of the freebase. FIG. 2F is aFTIR spectrum of the freebase. FIG. 2G is a ¹H NMR spectrum of thefreebase in DMSO-d⁶.

FIG. 3 is a differential scanning calorimetry scan of the maleic acidsalt of the compound of Formula I identified in the initial salt screen.

FIG. 4 is a differential scanning calorimetry scan of the acetic acidsalt of the compound of Formula I identified in the initial salt screen.

FIG. 5 is a differential scanning calorimetry scan of the D-malic acidsalt of the compound of Formula I identified in the initial salt screen.

FIG. 6 is a differential scanning calorimetry scan of the benzoic acidsalt of the compound of Formula I identified in the initial salt screen.

FIG. 7 is a differential scanning calorimetry scan of the L-tartaricacid salt of the compound of Formula I identified in the initial saltscreen.

FIG. 8 is a differential scanning calorimetry scan of the citric acidsalt of the compound of Formula I identified in the initial salt screen.

FIG. 9 is a differential scanning calorimetry scan of the propionic acidsalt of the compound of Formula I identified in the initial salt screen.

FIG. 10 is a differential scanning calorimetry scan of the D-tartaricacid salt of the compound of Formula I identified in the initial saltscreen.

FIG. 11 is a differential scanning calorimetry scan of the L-malic acidsalt of the compound of Formula I identified in the initial salt screen.

FIG. 12 is a differential scanning calorimetry scan of the sulfuric acidsalt of the compound of Formula I identified in the initial salt screen.

FIG. 13 is a differential scanning calorimetry scan of the hydrochloricacid salt of the compound of Formula I prepared on approximately 0.2 gscale.

FIG. 14 is a differential scanning calorimetry scan of the hydrobromicacid salt of the compound of Formula I prepared on approximately 0.2 gscale.

FIGS. 15A-15B are differential scanning calorimetry scans of thehydrochloric acid salt of the compound of Formula I prepared during theoptimization process. FIG. 15A is the differential scanning calorimetryscan of the hydrochloric acid salt (prepared using DMA as the solvent).FIG. 15B is the differential scanning calorimetry scan of thehydrochloric acid salt (prepared using a mixture of DCM/EtOH) as thesolvent.

FIGS. 16A-16F are scans of the hydrochloric acid salt of the compound ofFormula I prepared on 2 gram scale. FIG. 16A is a differential scanningcalorimetry scan of the hydrochloric acid salt (prepared using DMA asthe solvent). FIG. 16B is a isothermic (25° C.) dynamic vapor sorptionscan of the hydrochloric acid salt (prepared using DMA as the solvent).FIG. 16C is a thermogravimetric analysis scan of the hydrochloric acidsalt (prepared using DMA as the solvent).

FIG. 16D is an overlay of a differential scanning calorimetry scan and athermogravimetric analysis scan of the hydrochloric acid salt (preparedusing a mixture of DCM/EtOH as the solvent). FIG. 16E is an overlay ofX-ray powder diffraction scans of the hydrochloric acid salt preparedusing DMA or a mixture of DCM/EtOH as the solvent on different scalesbefore and after DVS. FIG. 16F is a ¹H NMR spectrum of the hydrochloricacid salt in DMSO-d⁶.

FIGS. 17A-17C are scans of the hydrobromic acid salt of the compound ofFormula I prepared on 2 gram scale. FIG. 17A is an overlay of adifferential scanning calorimetry scan and a thermogravimetric analysisscan of the hydrobromic acid salt. FIG. 17B is an X-ray powderdiffraction scan of the hydrobromic acid salt. FIG. 17C is a FTIRspectrum of the hydrobromic acid salt. FIG. 17D is a ¹H NMR spectrum ofthe hydrobromic acid salt in DMSO-d⁶.

FIGS. 18A-18H are scans of the L- and D-malic acid salts of the compoundof Formula I. FIG. 18A is an overlay of a differential scanningcalorimetry scan and a thermogravimetric analysis scan of the L-malicacid salt. FIG. 18B is an overlay of a differential scanning calorimetryscan and a thermogravimetric analysis scan of the D-malic acid salt.FIG. 18C is an isothermic (25° C.) dynamic vapor sorption scan of theL-malic salt. FIG. 18D is an isothermic (25° C.) dynamic vapor sorptionscan of the D-malic acid salt. FIG. 18E is an overlay of X-ray powderdiffraction scans of the L- and D-malic acid salts. FIG. 18F is anoverlay of FTIR spectra of the L- and D-malic acid salts. FIG. 18G is a¹H NMR spectrum of the malic acid salt in DMSO-d⁶. FIG. 18H is a ¹H NMRspectrum of the malic acid salt in DMSO-d⁶. FIGS. 18I-M are scans of twolots of the L-malic acid salt of the compound of Formula I. FIG. 18I isa differential scanning calorimetry scan of the L-malic acid salt (LotA). FIG. 18J is a thermogravimetric analysis scan of the L-malic acidsalt (Lot A). FIG. 18K is a differential scanning calorimetry scan ofthe L-malic acid salt (Lot B). FIG. 18L is a thermogravimetric analysisscan of the L-malic acid salt (Lot B). FIG. 18M is an overlay of X-raypowder diffraction scans of the L-malic acid salts (Lot A and Lot B) andthe freebase of the compound of Formula I.

FIGS. 19A-19F are scans of polymorph Form 1 of the compound of FormulaII. FIG. 19A is an X-ray powder diffraction scan of fully dried Form 1.FIG. 19B is a differential scanning calorimetry scan of Form 1. FIG. 19Cis a thermogravimetric/differential thermal analysis scan of Form 1.FIG. 19D is a gravimetric vapor sorption isotherm of Form 1. FIG. 19E isa kinetic gravimetric vapor sorption scan of Form 1. FIG. 19F is a ¹HNMR spectrum of Form 1 in d₆-DMSO.

FIGS. 20A-20E are scans of polymorph Form 2 of the compound of FormulaII. FIG. 20A shows X-ray powder diffraction scans of Form 2 (small scaleslurry, large scale slurry, and fully dried). FIG. 20B is a differentialscanning calorimetry scan of Form 2. FIG. 20C is athermogravimetric/differential thermal analysis scan of Form 2. FIG. 20Dis a gravimetric vapor sorption isotherm of Form 2. FIG. 20E is akinetic gravimetric vapor sorption scan of Form 2.

FIGS. 21A-21F are scans of polymorph Form 7 of the compound of FormulaII. FIG. 21A shows X-ray powder diffraction scans of Form 7 (small scaleslurry, large scale slurry, and fully dried). FIG. 21B is a differentialscanning calorimetry scan of Form 7. FIG. 21C is athermogravimetric/differential thermal analysis scan of Form 7. FIG. 21Dis a gravimetric vapor sorption isotherm of Form 7. FIG. 21E is akinetic gravimetric vapor sorption scan of Form 7. FIG. 21F is a ¹H NMRspectrum of Form 7 in d₆-DMSO.

FIGS. 22A-22F are scans of polymorph Form 8 of the compound of FormulaII. FIG. 22A shows X-ray powder diffraction scans of Form 8 (small scaleslurry, large scale slurry, and fully dried). FIG. 22B is a differentialscanning calorimetry scan of Form 8. FIG. 22C is athermogravimetric/differential thermal analysis scan of Form 8. FIG. 22Dis a gravimetric vapor sorption isotherm of Form 8. FIG. 22E is akinetic gravimetric vapor sorption scan of Form 8. FIG. 22F is a ¹H NMRspectrum of Form 8 in d₆-DMSO.

FIGS. 23A-23F are scans of the phosphate salt of the compound of FormulaII. FIG. 23A is an X-ray powder diffraction scan of the fully driedphosphate salt. FIG. 23B is a differential scanning calorimetry scan ofthe phosphate salt. FIG. 23C is a thermogravimetric/differential thermalanalysis scan of the phosphate salt. FIG. 23D is a gravimetric vaporsorption isotherm of the phosphate salt. FIG. 23E is a kineticgravimetric vapor sorption scan of the phosphate salt. FIG. 23F is a ¹HNMR spectrum of Form 1 in d₆-DMSO.

FIGS. 24A-24B are X-ray powder diffraction scans from a salt screen ofthe compound of Formula II with hydrochloric acid. FIG. 24A shows thescans of the compound of Formula II in each solvent tested after thesamples were temperature cycled between room temperature and 40° C. in4-hour cycles over 24 hours (post-cycling). FIG. 24B shows the scans ofthe compound of Formula II in each solvent tested after overnightstorage of the samples in an oven at 40° C. and 75% relative humidity(post-stability).

FIGS. 25A-25B are X-ray powder diffraction scans from a salt screen ofthe compound of Formula II with sulfuric acid. FIG. 25A shows the scansof the compound of Formula II in each solvent tested after the sampleswere temperature cycled between room temperature and 40° C. in 4-hourcycles over 24 hours (post-cycling). FIG. 25B shows the scans of thecompound of Formula II in each solvent tested after overnight storage ofthe samples in an oven at 40° C. and 75% relative humidity(post-stability).

FIGS. 26A-26B are X-ray powder diffraction scans from a salt screen ofthe compound of Formula II with p-toluene sulfonic acid. FIG. 26A showsthe scans of the compound of Formula II in each solvent tested after thesamples were temperature cycled between room temperature and 40° C. in4-hour cycles over 24 hours (post-cycling). FIG. 26B shows the scans ofthe compound of Formula II in each solvent tested after overnightstorage of the samples in an oven at 40° C. and 75% relative humidity(post-stability).

FIGS. 27A-27B are X-ray powder diffraction scans from a salt screen ofthe compound of Formula II with methane sulfonic acid. FIG. 27A showsthe scans of the compound of Formula II in each solvent tested after thesamples were temperature cycled between room temperature and 40° C. in4-hour cycles over 24 hours (post-cycling). FIG. 27B shows the scans ofthe compound of Formula II in each solvent tested after overnightstorage of the samples in an oven at 40° C. and 75% relative humidity(post-stability).

FIGS. 28A-28B are X-ray powder diffraction scans from a salt screen ofthe compound of Formula II with naphthalene-2-sulfonic acid. FIG. 28Ashows the scans of the compound of Formula II in each solvent testedafter the samples were temperature cycled between room temperature and40° C. in 4-hour cycles over 24 hours (post-cycling). FIG. 28B shows thescans of the compound of Formula II in each solvent tested afterovernight storage of the samples in an oven at 40° C. and 75% relativehumidity (post-stability).

FIGS. 29A-29B are X-ray powder diffraction scans from a salt screen ofthe compound of Formula II with benzene sulfonic acid. FIG. 29A showsthe scans of the compound of Formula II in each solvent tested after thesamples were temperature cycled between room temperature and 40° C. in4-hour cycles over 24 hours (post-cycling). FIG. 29B shows the scans ofthe compound of Formula II in each solvent tested after overnightstorage of the samples in an oven at 40° C. and 75% relative humidity(post-stability).

FIGS. 30A-30B are X-ray powder diffraction scans from a salt screen ofthe compound of Formula II with oxalic acid. FIG. 30A shows the scans ofthe compound of Formula II in each solvent tested after the samples weretemperature cycled between room temperature and 40° C. in 4-hour cyclesover 24 hours (post-cycling). FIG. 30B shows the scans of the compoundof Formula II in each solvent tested after overnight storage of thesamples in an oven at 40° C. and 75% relative humidity (post-stability).

FIGS. 31A-31B are X-ray powder diffraction scans from a salt screen ofthe compound of Formula II with 2-hydroxyethanesulfonic acid. FIG. 31Ashows the scans of the compound of Formula II in each solvent testedafter the samples were temperature cycled between room temperature and40° C. in 4-hour cycles over 24 hours (post-cycling). FIG. 31B shows thescans of the compound of Formula II in each solvent tested afterovernight storage of the samples in an oven at 40° C. and 75% relativehumidity (post-stability).

FIGS. 32A-32B are X-ray powder diffraction scans from a salt screen ofthe compound of Formula II with L-aspartic acid. FIG. 32A shows thescans of the compound of Formula II in each solvent tested after thesamples were temperature cycled between room temperature and 40° C. in4-hour cycles over 24 hours (post-cycling). FIG. 32B shows the scans ofthe compound of Formula II in each solvent tested after overnightstorage of the samples in an oven at 40° C. and 75% relative humidity(post-stability).

FIGS. 33A-33B are X-ray powder diffraction scans from a salt screen ofthe compound of Formula II with maleic acid. FIG. 33A shows the scans ofthe compound of Formula II in each solvent tested after the samples weretemperature cycled between room temperature and 40° C. in 4-hour cyclesover 24 hours (post-cycling). FIG. 33B shows the scans of the compoundof Formula II in each solvent tested after overnight storage of thesamples in an oven at 40° C. and 75% relative humidity (post-stability).

FIGS. 34A-34B are X-ray powder diffraction scans from a salt screen ofthe compound of Formula II with phosphoric acid. FIG. 34A shows thescans of the compound of Formula II in each solvent tested after thesamples were temperature cycled between room temperature and 40° C. in4-hour cycles over 24 hours (post-cycling). FIG. 34B shows the scans ofthe compound of Formula II in each solvent tested after overnightstorage of the samples in an oven at 40° C. and 75% relative humidity(post-stability).

FIGS. 35A-35B are X-ray powder diffraction scans from a salt screen ofthe compound of Formula II with ethanesulfonic acid. FIG. 35A shows thescans of the compound of Formula II in each solvent tested after thesamples were temperature cycled between room temperature and 40° C. in4-hour cycles over 24 hours (post-cycling). FIG. 35B shows the scans ofthe compound of Formula II in each solvent tested after overnightstorage of the samples in an oven at 40° C. and 75% relative humidity(post-stability).

FIGS. 36A-36B are X-ray powder diffraction scans from a salt screen ofthe compound of Formula II with L-glutamic acid. FIG. 36A shows thescans of the compound of Formula II in each solvent tested after thesamples were temperature cycled between room temperature and 40° C. in4-hour cycles over 24 hours (post-cycling). FIG. 36B shows the scans ofthe compound of Formula II in each solvent tested after overnightstorage of the samples in an oven at 40° C. and 75% relative humidity(post-stability).

FIGS. 37A-37B are X-ray powder diffraction scans from a salt screen ofthe compound of Formula II with L-tartaric acid. FIG. 37A shows thescans of the compound of Formula II in each solvent tested after thesamples were temperature cycled between room temperature and 40° C. in4-hour cycles over 24 hours (post-cycling). FIG. 37B shows the scans ofthe compound of Formula II in each solvent tested after overnightstorage of the samples in an oven at 40° C. and 75% relative humidity(post-stability).

FIGS. 38A-38B are X-ray powder diffraction scans from a salt screen ofthe compound of Formula II with fumaric acid. FIG. 38A shows the scansof the compound of Formula II in each solvent tested after the sampleswere temperature cycled between room temperature and 40° C. in 4-hourcycles over 24 hours (post-cycling). FIG. 38B shows the scans of thecompound of Formula II in each solvent tested after overnight storage ofthe samples in an oven at 40° C. and 75% relative humidity(post-stability).

FIGS. 39A-39B are X-ray powder diffraction scans from a salt screen ofthe compound of Formula II with citric acid. FIG. 39A shows the scans ofthe compound of Formula II in each solvent tested after the samples weretemperature cycled between room temperature and 40° C. in 4-hour cyclesover 24 hours (post-cycling). FIG. 39B shows the scans of the compoundof Formula II in each solvent tested after overnight storage of thesamples in an oven at 40° C. and 75% relative humidity (post-stability).

FIGS. 40A-40B are X-ray powder diffraction scans from a salt screen ofthe compound of Formula II with D-glucuronic acid. FIG. 40A shows thescans of the compound of Formula II in each solvent tested after thesamples were temperature cycled between room temperature and 40° C. in4-hour cycles over 24 hours (post-cycling). FIG. 40B shows the scans ofthe compound of Formula II in each solvent tested after overnightstorage of the samples in an oven at 40° C. and 75% relative humidity(post-stability).

FIGS. 41A-41B are X-ray powder diffraction scans from a salt screen ofthe compound of Formula II with L-malic acid. FIG. 41A shows the scansof the compound of Formula II in each solvent tested after the sampleswere temperature cycled between room temperature and 40° C. in 4-hourcycles over 24 hours (post-cycling). FIG. 41B shows the scans of thecompound of Formula II in each solvent tested after overnight storage ofthe samples in an oven at 40° C. and 75% relative humidity(post-stability).

FIGS. 42A-42B are X-ray powder diffraction scans from a salt screen ofthe compound of Formula II with hippuric acid. FIG. 42A shows the scansof the compound of Formula II in each solvent tested after the sampleswere temperature cycled between room temperature and 40° C. in 4-hourcycles over 24 hours (post-cycling). FIG. 42B shows the scans of thecompound of Formula II in each solvent tested after overnight storage ofthe samples in an oven at 40° C. and 75% relative humidity(post-stability).

FIGS. 43A-43B are X-ray powder diffraction scans from a salt screen ofthe compound of Formula II with D-gluconic acid. FIG. 43A shows thescans of the compound of Formula II in each solvent tested after thesamples were temperature cycled between room temperature and 40° C. in4-hour cycles over 24 hours (post-cycling). FIG. 43B shows the scans ofthe compound of Formula II in each solvent tested after overnightstorage of the samples in an oven at 40° C. and 75% relative humidity(post-stability).

FIGS. 44A-44B are X-ray powder diffraction scans from a salt screen ofthe compound of Formula II with L-lactic acid. FIG. 44A shows the scansof the compound of Formula II in each solvent tested after the sampleswere temperature cycled between room temperature and 40° C. in 4-hourcycles over 24 hours (post-cycling). FIG. 44B shows the scans of thecompound of Formula II in each solvent tested after overnight storage ofthe samples in an oven at 40° C. and 75% relative humidity(post-stability).

FIGS. 45A-45B are X-ray powder diffraction scans from a salt screen ofthe compound of Formula II with L-ascorbic acid. FIG. 45A shows thescans of the compound of Formula II in each solvent tested after thesamples were temperature cycled between room temperature and 40° C. in4-hour cycles over 24 hours (post-cycling). FIG. 45B shows the scans ofthe compound of Formula II in each solvent tested after overnightstorage of the samples in an oven at 40° C. and 75% relative humidity(post-stability).

FIGS. 46A-46B are X-ray powder diffraction scans from a salt screen ofthe compound of Formula II with benzoic acid. FIG. 46A shows the scansof the compound of Formula II in each solvent tested after the sampleswere temperature cycled between room temperature and 40° C. in 4-hourcycles over 24 hours (post-cycling). FIG. 46B shows the scans of thecompound of Formula II in each solvent tested after overnight storage ofthe samples in an oven at 40° C. and 75% relative humidity(post-stability).

FIGS. 47A-47B are X-ray powder diffraction scans from a salt screen ofthe compound of Formula II with succinic acid. FIG. 47A shows the scansof the compound of Formula II in each solvent tested after the sampleswere temperature cycled between room temperature and 40° C. in 4-hourcycles over 24 hours (post-cycling). FIG. 47B shows the scans of thecompound of Formula II in each solvent tested after overnight storage ofthe samples in an oven at 40° C. and 75% relative humidity(post-stability).

FIG. 48 is a thermogravimetric/differential thermal analysis scan of thesulfate salt of the compound of Formula II identified in the primarysalt screen.

FIG. 49 is a thermogravimetric/differential thermal analysis scan of thetosylate salt of the compound of Formula II identified in the primarysalt screen.

FIG. 50 is a thermogravimetric/differential thermal analysis scan of thenaphthalene-2-sulfonate salt of the compound of Formula II identified inthe primary salt screen.

FIG. 51 is a thermogravimetric/differential thermal analysis scan of theoxalate salt (1,4-dioxane/10% water) of the compound of Formula IIidentified in the primary salt screen.

FIG. 52 is a thermogravimetric/differential thermal analysis scan of theoxalate salt (evaporation) of the compound of Formula II identified inthe primary salt screen.

FIG. 53 is a thermogravimetric/differential thermal analysis scan of thephosphate salt (acetone/10% water) of the compound of Formula IIidentified in the primary salt screen.

FIG. 54 is a thermogravimetric/differential thermal analysis scan of thephosphate salt (IPA/10% water) of the compound of Formula II identifiedin the primary salt screen.

FIG. 55 is a thermogravimetric/differential thermal analysis scan of thetartrate salt of the compound of Formula II identified in the primarysalt screen.

FIG. 56 is a thermogravimetric/differential thermal analysis scan of thefumarate salt of the compound of Formula II identified in the primarysalt screen.

FIG. 57 shows X-ray powder diffraction scans of the observed solids fromthe solvents tested in the solvent solubility screen of Form I ofFormula II.

FIGS. 58A-58D show X-ray powder diffraction scans of the compound ofFormula II after temperature cycling experiments in various solvents andstorage at 40° C. and 75% RH overnight.

FIG. 59 shows X-ray powder diffraction scans of the compound of FormulaII after evaporation experiments using various solvents.

FIG. 60 shows X-ray powder diffraction scans of the compound of FormulaII after crash-cooling experiments using various solvents.

FIG. 61 shows X-ray powder diffraction scans of the compound of FormulaII after anti-solvent experiments using various solvents.

FIGS. 62A-62F are scans of the compound of Formula II. FIG. 62A is anX-ray powder diffraction scan of the compound of Formula II. FIG. 62B isa differential scanning calorimetry scan of the compound of Formula II.FIG. 62C is a thermogravimetric/differential thermal analysis scan ofthe compound of Formula II. FIG. 62D is a dynamic vapor sorptionisotherm of the compound of Formula II. FIG. 62E is a kinetic dynamicvapor sorption scan of the compound of Formula II. FIG. 62F is a ¹H NMRspectrum of the compound of Formula II in d₆-DMSO.

FIGS. 63A-63B are scans of the polymorph Form A of the compound ofFormula III. FIG. 63A is an X-ray powder diffraction scan of thepolymorph Form A of the compound of Formula III. FIG. 63B is adifferential scanning calorimetry scan of the polymorph Form A of thecompound of Formula III.

FIGS. 64A-64B are the scans of polymorph Form A of the compound ofFormula IV. FIG. 64A is an X-ray powder diffraction scan of polymorphForm A of the compound of Formula IV. FIG. 64B is a differentialscanning calorimetry scan of polymorph Form A of the compound of FormulaIV.

FIGS. 65A-B are the scans of polymorph Form B of the compound of FormulaIV. FIG. 65A is an X-ray powder diffraction scan of polymorph Form B ofthe compound of Formula IV. FIG. 65B is a differential scanningcalorimetry scan of polymorph Form B of the compound of Formula IV.

FIG. 66 is an overlay of the X-ray powder diffraction scans ofpolymorphs A and B of the compound of Formula IV.

DETAILED DESCRIPTION 1. Definitions

The term “polymorph,” as used herein, refers to crystals of the samecompound having different physical properties as a result of the orderof the molecules in the crystal lattice. Different polymorphs of asingle compound have one or more different chemical, physical,mechanical, electrical, thermodynamic, and/or biological properties fromeach other. Differences in physical properties exhibited by polymorphscan affect pharmaceutical parameters such as storage stability,compressibility, density (important in composition and productmanufacturing), dissolution rates (an important factor in determiningbio-availability), solubility, melting point, chemical stability,physical stability, powder flowability, water sorption, compaction, andparticle morphology. Differences in stability can result from changes inchemical reactivity (e.g., differential oxidation, such that a dosageform discolors more rapidly when comprised of one polymorph than whencomprised of another polymorph) or mechanical changes (e.g., crystalchanges on storage as a kinetically favored polymorph converts to athermodynamically more stable polymorph) or both (e.g., one polymorph ismore hygroscopic than the other). As a result of solubility/dissolutiondifferences, some transitions affect potency and/or toxicity. Inaddition, the physical properties of the crystal may be important inprocessing; for example, one polymorph might be more likely to formsolvates or might be difficult to filter and wash free of impurities(i.e., particle shape and size distribution might be different betweenone polymorph relative to the other). “Polymorph”, as used herein, doesnot include amorphous forms of the compound. As used herein, “amorphous”refers to a noncrystalline form of a compound which can be a solid stateform of the compound or a solubilized form of the compound. For example,“amorphous” refers to a compound (e.g., a solid form of the compound)without a regularly repeating arrangement of molecules or external faceplanes.

The term “anhydrous,” as used herein, refers to a crystal form of thecompound of Formula I-IV that has 1% or less by weight water. Forexample, 0.5% or less, 0.25% or less, or 0.1% or less by weight water.

The term “solvate” as used herein refers to a crystalline form of acompound of Formula I-IV, such as a polymorph form of the compound,where the crystal lattice comprises one or more solvents ofcrystallization.

The terms “hydrate” or “hydrated polymorph form” refer to a crystallineform of a compound of Formula I-IV, such as a polymorph form of thecompound, where the crystal lattice comprises water. Unless specifiedotherwise, the term “hydrate” as used herein refers to a “stoichiometrichydrate.” A stoichiometric hydrate contains the water molecules as anintegral part of the crystal lattice, where removal of the watermolecules will cause instability of the crystal network. In comparison,a non-stoichiometric hydrate comprises water, but changes in the watercontent does not cause significant changes to the crystal structure.During drying of non-stoichiometric hydrates, a considerable proportionof water can be removed without significantly disturbing the crystalnetwork, and the crystals can subsequently rehydrate to give the initialnon-stoichiometric hydrated crystalline form. Unlike stoichiometrichydrates, the dehydration and rehydration of non-stoichiometric hydratesis not accompanied by a phase transition, and thus all hydration statesof a non-stoichiometric hydrate represent the same crystal form.

“Purity,” when used in reference to a composition including a polymorphof a compound of Formula I-IV, refers to the percentage of one specificpolymorph form relative to another polymorph form or an amorphous formof a compound of Formula I-IV in the referenced composition. Forexample, a composition comprising polymorph Form 1 having a purity of90% would comprise 90 weight parts Form 1 and 10 weight parts of otherpolymorph and/or amorphous forms of the compound of Formula I-IV.

As used herein, a compound or composition is “substantially free of” oneor more other components if the compound or composition contains nosignificant amount of such other components. For example, thecomposition can contain less than 5%, 4%, 3%, 2%, or 1% by weight ofother components. Such components can include starting materials,residual solvents, or any other impurities that can result from thepreparation of and/or isolation of the compounds and compositionsprovided herein. In some embodiments, a polymorph form provided hereinis substantially free of other polymorph forms. In some embodiments, aparticular polymorph of the compound of Formula I-IV is “substantiallyfree” of other polymorphs if the particular polymorph constitutes atleast about 95% by weight of the compound of Formula I-IV present. Insome embodiments, a particular polymorph of the compound of Formula I-IVis “substantially free” of other polymorphs if the particular polymorphconstitutes at least about 97%, about 98%, about 99%, or about 99.5% byweight of the compound of Formula I-IV present. In certain embodiments,a particular polymorph of the compound of Formula I-IV is “substantiallyfree” of water if the amount of water constitutes no more than about 2%,about 1%, or about 0.5% by weight of the polymorph.

As used herein, “substantially pure,” when used in reference to apolymorph form of the compound of Formula I-IV, means a sample of apolymorph form of the compound having a purity greater than 90%,including greater than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and99%, and also including equal to about 100% of the compound, based onthe weight of the compound. The remaining material comprises otherform(s) of the compound, and/or reaction impurities and/or processingimpurities arising from its preparation. For example, a polymorph formof the compound of Formula I-IV may be deemed substantially pure in thatit has a purity greater than 90% of a polymorph form of the compound ofFormula I-IV, as measured by means that are at this time known andgenerally accepted in the art, where the remaining less than 10% ofmaterial comprises other form(s) of the compound of Formula I-IV and/orreaction impurities and/or processing impurities. The presence ofreaction impurities and/or processing impurities may be determined byanalytical techniques known in the art, such as, for example,chromatography, nuclear magnetic resonance spectroscopy, massspectrometry, or infrared spectroscopy.

The term “about” preceding a value for DSC, TGA, TG, or DTA, which arereported as degrees Celsius, have an allowable variability of ±5° C.

To provide a more concise description, some of the quantitativeexpressions herein are recited as a range from about amount X to aboutamount Y. It is understood that when a range is recited, the range isnot limited to the recited upper and lower bounds, but rather includesthe full range from about amount X through about amount Y, or any rangetherein.

“Room temperature” or “RT” refers to the ambient temperature of atypical laboratory, which is typically around 25° C.

As used herein, the terms “subject,” “individual,” or “patient,” usedinterchangeably, refer to any animal, including mammals such as mice,rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses,primates, and humans. In some embodiments, the patient is a human. Insome embodiments, the subject has experienced and/or exhibited at leastone symptom of the disease or disorder to be treated and/or prevented.In some embodiments, the subject has been identified or diagnosed ashaving a cancer with dysregulation of a RET gene, a RET protein, orexpression or activity, or level of any of the same (a RET-associatedcancer) (e.g., as determined using a regulatory agency-approved, e.g.,FDA-approved, assay or kit). In some embodiments, the subject has atumor that is positive for dysregulation of a RET gene, a RET protein,or expression or activity, or level of any of the same (e.g., asdetermined using a regulatory agency-approved assay or kit). The subjectcan be a subject with a tumor(s) that is positive for dysregulation of aRET gene, a RET protein, or expression or activity, or level of any ofthe same (e.g., identified as positive using a regulatoryagency-approved, e.g., FDA-approved, assay or kit). The subject can be asubject whose tumors have dysregulation of a RET gene, a RET protein, orexpression or activity, or a level of the same (e.g., where the tumor isidentified as such using a regulatory agency-approved, e.g.,FDA-approved, kit or assay). In some embodiments, the subject issuspected of having a RET-associated cancer. In some embodiments, thesubject has a clinical record indicating that the subject has a tumorthat has dysregulation of a RET gene, a RET protein, or expression oractivity, or level of any of the same (and optionally the clinicalrecord indicates that the subject should be treated with any of thecompositions provided herein). In some embodiments, the patient is apediatric patient.

The term “pediatric patient” as used herein refers to a patient underthe age of 21 years at the time of diagnosis or treatment. The term“pediatric” can be further divided into various subpopulationsincluding: neonates (from birth through the first month of life);infants (1 month up to two years of age); children (two years of age upto 12 years of age); and adolescents (12 years of age through 21 yearsof age (up to, but not including, the twenty-second birthday)). BerhmanR E, Kliegman R, Arvin A M, Nelson W E, Textbook of Pediatrics, 15th Ed.Philadelphia: W.B. Saunders Company, 1996; Rudolph A M, et al.,Rudolph's Pediatrics, 21st Ed. New York: McGraw-Hill, 2002; and Avery MD, First L R, Pediatric Medicine, 2nd Ed. Baltimore: Williams & Wilkins;1994. In some embodiments, a pediatric patient is from birth through thefirst 28 days of life, from 29 days of age to less than two years ofage, from two years of age to less than 12 years of age, or 12 years ofage through 21 years of age (up to, but not including, the twenty-secondbirthday). In some embodiments, a pediatric patient is from birththrough the first 28 days of life, from 29 days of age to less than 1year of age, from one month of age to less than four months of age, fromthree months of age to less than seven months of age, from six months ofage to less than 1 year of age, from 1 year of age to less than 2 yearsof age, from 2 years of age to less than 3 years of age, from 2 years ofage to less than seven years of age, from 3 years of age to less than 5years of age, from 5 years of age to less than 10 years of age, from 6years of age to less than 13 years of age, from 10 years of age to lessthan 15 years of age, or from 15 years of age to less than 22 years ofage.

As used herein, the terms “treat” or “treatment” refer to therapeutic orpalliative measures. Beneficial or desired clinical results include, butare not limited to, alleviation, in whole or in part, of symptomsassociated with a disease or disorder or condition, diminishment of theextent of disease, stabilized (i.e., not worsening) state of disease,delay or slowing of disease progression, amelioration or palliation ofthe disease state (e.g., one or more symptoms of the disease), andremission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment.

The term “administration” or “administering” refers to a method ofgiving a dosage of a compound or pharmaceutical composition to avertebrate or invertebrate, including a mammal, a bird, a fish, or anamphibian. The preferred method of administration can vary depending onvarious factors, e.g., the components of the pharmaceutical composition,the site of the disease, and the severity of the disease.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” includes any and all solvents, co-solvents,complexing agents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, which are not biologically or otherwise undesirable. The use ofsuch media and agents for pharmaceutically active substances iswell-known in the art. Except insofar as any conventional media or agentis incompatible with the active ingredient, its use in the therapeuticcompositions provided herein is contemplated. Supplementary activeingredients can also be incorporated into the compositions. In addition,various excipients, such as are commonly used in the art, can beincluded. These and other such compounds are described in theliterature, e.g., in the Merck Index, Merck & Company, Rahway, N.J.Considerations for the inclusion of various components in pharmaceuticalcompositions are described, e.g., in Gilman et al. (Eds.) (2010);Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 12thEd., The McGraw-Hill Companies.

By “therapeutically effective amount” or “pharmaceutically effectiveamount” of a compound as provided herein is an amount which issufficient to achieve the desired effect and can vary according to thenature and severity of the disease condition, and the potency of thecompound. A therapeutic effect is the relief, to some extent, of one ormore of the symptoms of the disease, and can include curing a disease.“Curing” means that the symptoms of active disease are eliminated.However, certain long-term or permanent effects of the disease can existeven after a cure is obtained (such as, e.g., extensive tissue damage).

The term “RET-associated disease or disorder” as used herein refers todiseases or disorders associated with or having a dysregulation of a RETgene, a RET kinase (also called herein RET kinase protein or RETkinase), or the expression or activity or level of any (e.g., one ormore) of the same (e.g., any of the types of dysregulation of a RETgene, a RET kinase, a RET kinase domain, or the expression or activityor level of any of the same described herein). Non-limiting examples ofa RET-associated disease or disorder include, for example, cancer andgastrointestinal disorders such as irritable bowel syndrome (IBS).

The term “RET-associated cancer” as used herein refers to cancersassociated with or having a dysregulation of a RET gene, a RET kinase(also called herein RET kinase protein or RET kinase), or expression oractivity, or level of any of the same. Non-limiting examples of aRET-associated cancer are described herein.

The phrase “dysregulation of a RET gene, a RET kinase, or the expressionor activity or level of any of the same” refers to a genetic mutation(e.g., a RET gene translocation that results in the expression of afusion protein, a deletion in a RET gene that results in the expressionof a RET protein that includes a deletion of at least one amino acid ascompared to the wild-type RET protein, or a mutation in a RET gene thatresults in the expression of a RET protein with one or more pointmutations, or an alternative spliced version of a RET mRNA that resultsin a RET protein that results in the deletion of at least one amino acidin the RET protein as compared to the wild-type RET protein), or a RETgene amplification that results in overexpression of a RET protein or anautocrine activity resulting from the overexpression of a RET gene acell, that results in a pathogenic increase in the activity of a kinasedomain of a RET protein (e.g., a constitutively active kinase domain ofa RET protein) in a cell. As another example, a dysregulation of a RETgene, a RET protein, or expression or activity, or level of any of thesame, can be a mutation in a RET gene that encodes a RET protein that isconstitutively active or has increased activity as compared to a proteinencoded by a RET gene that does not include the mutation. For example, adysregulation of a RET gene, a RET protein, or expression or activity,or level of any of the same, can be the result of a gene or chromosometranslocation which results in the expression of a fusion protein thatcontains a first portion of RET that includes a functional kinasedomain, and a second portion of a partner protein (i.e., that is notRET). In some examples, dysregulation of a RET gene, a RET protein, orexpression or activity, can be a result of a gene translocation of oneRET gene with another non-RET gene. Non-limiting examples of fusionproteins are described in Table 1. Non-limiting examples of RET kinaseprotein point mutations/insertions/deletions are described in Table 2.Additional examples of RET kinase protein mutations (e.g., pointmutations) are RET inhibitor resistance mutations. Non-limiting examplesof RET inhibitor resistance mutations are described in Tables 3 and 4.

The term “wildtype” or “wild-type” describes a nucleic acid (e.g., a RETgene or a RET mRNA) or protein (e.g., a RET protein) that is found in asubject that does not have a RET-associated disease, e.g., aRET-associated cancer (and optionally also does not have an increasedrisk of developing a RET-associated disease and/or is not suspected ofhaving a RET-associated disease), or is found in a cell or tissue from asubject that does not have a RET-associated disease, e.g., aRET-associated cancer (and optionally also does not have an increasedrisk of developing a RET-associated disease and/or is not suspected ofhaving a RET-associated disease).

The term “regulatory agency” refers to a country's agency for theapproval of the medical use of pharmaceutical agents with the country.For example, a non-limiting example of a regulatory agency is the U.S.Food and Drug Administration (FDA).

2. Polymorphs and Pharmaceutically Acceptable Salts

The present disclosure relates to compounds of Formula I-IV andpharmaceutically acceptable salts thereof which exhibit rearrangedduring transfection (RET) kinase inhibition. In particular, providedherein are novel crystalline forms of4-(6-(4-((6-methoxypyridin-3-yl)methyl)piperazin-1-yl)pyridin-3-yl)-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile(Formula I);6-(2-hydroxy-2-methylpropoxy)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile(Formula II);6-(2-hydroxy-2-methylpropoxy)-4-(6-(6-(6-methoxynicotinoyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile(Formula III); and6-(2-hydroxy-2-methylpropoxy)-4-(6-(4-hydroxy-4-(pyridin-2-ylmethyl)piperidin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile(Formula IV), and pharmaceutically acceptable salts thereof,pharmaceutical compositions comprising the compounds, processes formaking the compounds, and the use of the compounds in therapy. Moreparticularly, it relates to novel crystalline forms of Formula I-IV andpharmaceutically acceptable salts thereof useful in the treatment andprevention of diseases which can be treated with a RET kinase inhibitor,including RET-associated diseases and disorders.

Formula I

Provided herein is a compound of Formula I:

including pharmaceutically acceptable salts, amorphous, and polymorphforms thereof.

The compound of Formula I provided herein can be prepared using methodsknown and understood by those of ordinary skill in the art. For example,synthetic methods such as those described in U.S. Publication No.2017/0096425 can be used, and this application is herein incorporated byreference in its entirety.

Provided herein are polymorph forms of the compound of Formula I. Theforms include, e.g., free bases, solvates, hydrates, salts, andnon-solvated forms of the compound of Formula I, including, for example,polymorph Form A. In some embodiments, the polymorph form of thecompound of Formula I is a pharmaceutically acceptable salt. In someembodiments, the compound of Formula I is a chloride salt. In someembodiments, the compound of Formula I is a bromide salt. In someembodiments, the compound of Formula I is an L-malate salt. In someembodiments, the compound of Formula I is a D-malate salt.

Form A

One such polymorph is a polymorph known as Form A. Form A is a polymorphform of the compound of Formula I. In some embodiments, Form A has anXRPD pattern, obtained with CuKα1-radiation, with at least peaks at °2θvalues of 4.4±0.2, 14.6±0.2, and 18.3±0.2. In some embodiments, Form Ahas an XRPD pattern with at least peaks at °2θ values of 4.4±0.2,13.5±0.2, 14.6±0.2, 18.3±0.2, and 18.8±0.2. In some embodiments, Form Ahas an XRPD pattern with at least peaks at °2θ values of 4.4±0.2,13.5±0.2, 14.6±0.2, 17.4±0.2, 18.3±0.2, 18.8±0.2, 21.0±0.2, and24.6±0.2. For example, in some embodiments, Form A has an XRPD patternwith at least peaks at °2θ values of 4.4±0.2, 13.5±0.2, 14.6±0.2,17.4±0.2, 18.3±0.2, 18.8±0.2, 21.0±0.2, 22.5±0.2, 24.6±0.2, and27.7±0.2.

In some embodiments, provided herein is a composition comprisingpolymorph Form A. In some embodiments, the composition can besubstantially pure. For example, the composition has a purity of atleast about 90%. In some embodiments, the composition has a purity of atleast about 95%. In some embodiments, the composition has a purity of atleast about 98%. For example, the composition can have a purity of atleast 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%,99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%. In some embodiments, thecomposition is substantially free of other forms of the compound ofFormula I. In some embodiments, the composition contains less than about15% by weight of other forms of the compound of Formula I. For example,the composition can contain less than 14%, 13%, 12%, 11%, 10%, 9%, 8%,7%, 6%, 5%, 4%, 3%, 2%, 1% by weight of one or more other forms of thecompound of Formula I. For example, the composition can contain lessthan about 15% of amorphous form.

In some embodiments, provided herein is polymorph Form A that exhibitsan endotherm that is observed between about 185-195° C., e.g., about192.8° C., as measured by DSC related to sorbed water. In someembodiments, provided herein is polymorph Form A that exhibits anendotherm that is observed between about 220-230° C., e.g., about 226.7°C., as measured by DSC related to sorbed water.

In some embodiments, provided herein is polymorph Form A that exhibits aweight loss of about 1.1% from the onset of heating to about 238° C., asmeasured by TGA.

Provided herein are methods of preparing polymorph Form A. In someembodiments, polymorph Form A of the compound of Formula I is preparedby contacting6-(-1-methyl-1H-pyrazol-4-yl)-4-(6-(piperazin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitriletetrahydrochloride, sodium triacetoxyborohydride, and triethylamine in apolar aprotic solvent. In some embodiments, the polar aprotic solvent isDMSO. In some embodiments, the method further comprises heating a slurrycomprising6-(-1-methyl-1H-pyrazol-4-yl)-4-(6-(piperazin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitriletetrahydrochloride, sodium triacetoxyborohydride, trimethylamine, andDMSO to about 30° C. In some embodiments, the method further comprisesheating the slurry for about 10 hours to about 15 hours, e.g., about 13hours. In some embodiments, the method further comprises cooling theslurry to about 19° C. after about 13 hours of heating at about 30° C.In some embodiments, the method further comprises adding water to theslurry. For example, the method can further comprise adding 2 volumes ofwater to the slurry. In same embodiments, the method comprises ageing acomposition comprising the slurry and water. In some embodiments, themethod comprises ageing a composition comprising the slurry and waterfor about 1 hour to about 10 hours, e.g., about 3.5 hours. In someembodiments, the method comprises isolating the solid throughfiltration. In some embodiments, the solid is dried. In someembodiments, the solid is dried under vacuum. In some embodiments, thesolid is dried at about 45° C.

Salts of Formula I

In some embodiments, the compound of Formula I is a pharmaceuticallyacceptable salt. For example, pharmaceutically acceptable salts of thecompound of Formula I can include, but are not limited to, chloride,bromide, sulfate, citrate, L-tartrate, D-tartrate, acetate, L-malate,D-malate, benzoate, propionate, and maleate salts. In some embodiments,the compound of Formula I is a chloride salt. In some embodiments, thechloride salt is prepared in a mixture of solvents. In some embodiments,the solvent is a mixture of dichloromethane and ethanol. In someembodiments, the ratio of dicholoromethane and ethanol is about 3.6:1 byvolume. In some embodiments, the ratio of dicholoromethane and ethanolis about 1:1 by volume. In some embodiments, the chloride salt isprepared in dimethylacetamide. In some embodiments, the compound ofFormula I is a bromide salt. In some embodiments, the bromide salt isprepared in a mixture of solvents. In some embodiments, the solvent is amixture of dichloromethane and ethanol. In some embodiments, the ratioof dicholoromethane and ethanol is about 3.6:1 by volume. In someembodiments, the bromide salt is prepared in dimethylacetamide. In someembodiments, the compound of Formula I is a sulfate salt. In someembodiments, the sulfate salt is prepared in a mixture of solvents. Insome embodiments, the sulfate salt is prepared in a mixture ofdichloromethane and ethanol. In some embodiments, the ratio ofdicholoromethane and ethanol is about 4:1 by volume. In someembodiments, the compound of Formula I is a citrate salt. In someembodiments, the citrate salt is prepared in dichloromethane. In someembodiments, the compound of Formula I is an L-tartrate salt. In someembodiments, the L-tartrate salt is prepared in dichloromethane. In someembodiments, the compound of Formula I is a D-tartrate salt. In someembodiments, the D-tartrate salt is prepared in dichloromethane. In someembodiments, the compound of Formula I is an acetate salt. In someembodiments, the acetate salt is prepared in dichloromethane. In someembodiments, the compound of Formula I is an L-malate salt. In someembodiments, the L-malate salt is prepared in dichloromethane. In someembodiments, the L-malate salt is prepared in a mixture of solvents. Insome embodiments, the L-malate salt is prepared in a mixture ofdichloromethane and ethanol. In some embodiments, the ratio ofdicholoromethane and ethanol is about 6:1 by volume. In someembodiments, the ratio of dicholoromethane and ethanol is about 3.6:1 byvolume. In some embodiments, the ratio of dicholoromethane and ethanolis about 3.3:1 by volume. In some embodiments, the compound of Formula Iis a D-malate salt. In some embodiments, the D-malate salt is preparedin dichloromethane. In some embodiments, the D-malate salt is preparedin a mixture of solvents. In some embodiments, the D-malate salt isprepared in a mixture of dichloromethane and ethanol. In someembodiments, the ratio of dicholoromethane and ethanol is about 3.6:1 byvolume. In some embodiments, the compound of Formula I is a benzoatesalt. In some embodiments, the benzoate salt is prepared indichloromethane. In some embodiments, the compound of Formula I is apropionate salt. In some embodiments, the propionate salt is prepared indichloromethane. In some embodiments, the compound of Formula I is amaleate salt. In some embodiments, the maleate salt is prepared indichloromethane.

Provided herein is a chloride salt of the compound of Formula I. In someembodiments, the chloride salt has a ratio of about 1.1:1, Cl:free base.

In some embodiments, provided herein is a composition comprising thechloride salt of the compound of Formula I. In some embodiments, thecomposition can be substantially pure. For example, the composition hasa purity of at least about 90%. In some embodiments, the composition hasa purity of at least about 95%. In some embodiments, the composition hasa purity of at least about 98%. For example, the composition can have apurity of at least 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%,99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%. In some embodiments,the composition is substantially free of other forms of the compound ofFormula I. In some embodiments, the composition contains less than about15% by weight of other forms of the compound of Formula I. For example,the composition can contain less than 14%, 13%, 12%, 11%, 10%, 9%, 8%,7%, 6%, 5%, 4%, 3%, 2%, 1% by weight of one or more other forms of thecompound of Formula I.

In some embodiments, provided herein is a chloride salt of the compoundof Formula I that exhibits an endotherm that is observed between about230-245° C., e.g., about 241° C. or 234° C., as measured by DSC relatedto sorbed water. In some embodiments, provided herein is a chloride saltof the compound of Formula I that exhibits a melting point of about 241°C., as measured by DSC.

In some embodiments, the chloride salt of the compound of Formula Iundergoes a mass loss of about 7.4% from the onset of heating to about255° C., as measured by TGA.

Provided herein are methods of preparing a chloride salt of the compoundof Formula I. In some embodiments, the method comprises slurrying acomposition comprising the compound of Formula I in a mixture ofdichloromethane and ethanol and adding a hydrochloric acid solution tothe mixture to generate the chloride salt as a residual solid. In someembodiments, the ratio of dicholoromethane and ethanol is about 3.6:1 byvolume. In some embodiments, the ratio of dicholoromethane and ethanolis about 1:1 by volume. In some embodiments, the method comprisesslurrying a composition comprising the compound of Formula I indimethylacetamide and adding a hydrochloric acid solution to the mixtureto generate the chloride salt as a residual solid. In some embodiments,the hydrochloric acid is added in a water solution. In some embodiments,the solvent is used in about 46 volumes. In some embodiments, thesolvent is used in about 50 volumes. In some embodiments, the solvent isused in about 75 volumes. In some embodiments, the slurry is temperaturecycled between about 0° C. and about RT. In some embodiments, the slurryis temperature cycled between about 30° C. and about RT. In someembodiments, the slurry is temperature cycled between about 40° C. andabout RT. In some embodiments, the method comprises adding MTBE to theslurry. In some embodiments, the method comprises adding about 125volumes of MTBE. In some embodiments, the method comprises ageing theslurry. In some embodiments, the method comprises ageing the slurry forabout 3 hours. In some embodiments, the method comprises ageing theslurry for about 13 hours. In some embodiments, the method comprisesageing the slurry for about 20 hours to about 40 hours, e.g., about 30hours. In some embodiments, the method comprises stirring the slurry. Insome embodiments, the method comprises isolating the solid throughfiltration.

Provided herein is a bromide salt of the compound of Formula I. In someembodiments, the bromide salt has a ratio of about 1.1:1, Br:free base.

In some embodiments, provided herein is a composition comprising thebromide salt of the compound of Formula I. In some embodiments, thecomposition can be substantially pure. For example, the composition hasa purity of at least about 90%. In some embodiments, the composition hasa purity of at least about 95%. In some embodiments, the composition hasa purity of at least about 98%. For example, the composition can have apurity of at least 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%,99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%. In some embodiments,the composition is substantially free of other forms of the compound ofFormula I. In some embodiments, the composition contains less than about15% by weight of other forms of the compound of Formula I. For example,the composition can contain less than 14%, 13%, 12%, 11%, 10%, 9%, 8%,7%, 6%, 5%, 4%, 3%, 2%, 1% by weight of one or more other forms of thecompound of Formula I.

In some embodiments, provided herein is a bromide salt of the compoundof Formula I that exhibits an endotherm that is observed between about235-250° C., e.g., about 238° C., as measured by DSC related to sorbedwater. In some embodiments, provided herein is a bromide salt of thecompound of Formula I that exhibits an endotherm that is observedbetween about 220-235° C., e.g., about 225° C., as measured by DSCrelated to sorbed water. In some embodiments, provided herein is abromide salt of the compound of Formula I that exhibits a melting pointof about 225° C., as measured by DSC. In some embodiments, providedherein is a bromide salt of the compound of Formula I that exhibits amelting point of about 238° C., as measured by DSC.

In some embodiments, the bromide salt of the compound of Formula Iundergoes a mass loss of about 10.3% from the onset of heating to about255° C., as measured by TGA.

Provided herein are methods of preparing a bromide salt of the compoundof Formula I. In some embodiments, the method comprises slurrying acomposition comprising the compound of Formula I in a mixture ofdichloromethane and ethanol and adding a hydrobromic acid solution tothe mixture to generate the bromide salt as a residual solid. In someembodiments, the ratio of dicholoromethane and ethanol is about 3.6:1 byvolume. In some embodiments, the method comprises slurrying acomposition comprising the compound of Formula I in dimethylacetamideand adding a hydrobromic acid solution to the mixture to generate thebromide salt as a residual solid. In some embodiments, the hydrobromicacid is added in a water solution. In some embodiments, the solvent isused in 46 volumes. In some embodiments, the solvent is used in about 50volumes. In some embodiments, the slurry is temperature cycled betweenaround about 0° C. and about RT. In some embodiments, the slurry istemperature cycled between around about 30° C. and about RT. In someembodiments, the slurry is temperature cycled between around about 40°C. and about RT. In some embodiments, the method comprises furthercomprises adding MTBE to the slurry. In some embodiments, the methodcomprises adding about 150 volumes of MTBE. In some embodiments, themethod comprises ageing the slurry. In some embodiments, the methodcomprises ageing the slurry for about 1 hour. In some embodiments, themethod comprises ageing the slurry for about 13 hours. In someembodiments, the method comprises ageing the slurry for about 10 hoursto about 30 hours, e.g., about 20 hours. In some embodiments, the methodcomprises isolating the solid through filtration.

Provided herein is an L-malate salt of the compound of Formula I. Insome embodiments, the L-malate salt has a ratio of about 0.97:1,malate:free base.

In some embodiments, provided herein is a composition comprising theL-malate salt of the compound of Formula I. In some embodiments, thecomposition can be substantially pure. For example, the composition hasa purity of at least about 90%. In some embodiments, the composition hasa purity of at least about 95%. In some embodiments, the composition hasa purity of at least about 98%. For example, the composition can have apurity of at least 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%,99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%. In some embodiments,the composition is substantially free of other forms of the compound ofFormula I. In some embodiments, the composition contains less than about15% by weight of other forms of the compound of Formula I. For example,the composition can contain less than 14%, 13%, 12%, 11%, 10%, 9%, 8%,7%, 6%, 5%, 4%, 3%, 2%, 1% by weight of one or more other forms of thecompound of Formula I.

In some embodiments, provided herein is an L-malate salt of the compoundof Formula I that exhibits an endotherm that is observed between about205-220° C., e.g., about 208° C., as measured by DSC related to sorbedwater. In some embodiments, provided herein is an L-malate salt of thecompound of Formula I that exhibits a melting point of about 208° C., asmeasured by DSC.

In some embodiments, the L-malate salt of the compound of Formula Iundergoes a mass loss of about 17.7% from the onset of heating to about253° C., as measured by TGA.

Provided herein are methods of preparing an L-malate salt of thecompound of Formula I. In some embodiments, the method comprisesslurrying a composition comprising the compound of Formula I in amixture of dichloromethane and ethanol and adding an L-malic acidsolution to the mixture to generate the L-malate salt as a residualsolid. In some embodiments, the ratio of dicholoromethane and ethanol isabout 3.6:1 by volume. In some embodiments, the ratio ofdicholoromethane and ethanol is about 3.3:1 by volume. In someembodiments, the ratio of dicholoromethane and ethanol is about 6:1 byvolume. In some embodiments, the L-malic acid is added in an ethanolsolution. In some embodiments, the solvent is used in about 46 volumes.In some embodiments, the solvent is used in about 26 volumes. In someembodiments, the slurry is temperature cycled between about 0° C. andabout RT. In some embodiments, the method comprises ageing the slurry.In some embodiments, the method comprises ageing the slurry for about 5hours to about 24 hours, e.g., about 13 hours. In some embodiments, themethod comprises ageing the slurry for about 10 hours to about 30 hours,e.g., about 20 hours. In some embodiments, the method comprises stirringthe slurry. In some embodiments, the method comprises isolating thesolid through filtration.

Provided herein is an D-malate salt of the compound of Formula I. Insome embodiments, the D-malate salt has a ratio of about 0.97:1,malate:free base.

In some embodiments, provided herein is a composition comprising theD-malate salt of the compound of Formula I. In some embodiments, thecomposition can be substantially pure. For example, the composition hasa purity of at least about 90%. In some embodiments, the composition hasa purity of at least about 95%. In some embodiments, the composition hasa purity of at least about 98%. For example, the composition can have apurity of at least 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%,99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%. In some embodiments,the composition is substantially free of other forms of the compound ofFormula I. In some embodiments, the composition contains less than about15% by weight of other forms of the compound of Formula I. For example,the composition can contain less than 14%, 13%, 12%, 11%, 10%, 9%, 8%,7%, 6%, 5%, 4%, 3%, 2%, 1% by weight of one or more other forms of thecompound of Formula I.

In some embodiments, provided herein is a D-malate salt of the compoundof Formula I that exhibits an endotherm that is observed between about205-215° C., e.g., about 208° C., as measured by DSC related to sorbedwater. In some embodiments, provided herein is an D-malate salt of thecompound of Formula I that exhibits a melting point of about 209° C., asmeasured by DSC.

In some embodiments, the D-malate salt of the compound of Formula Iundergoes a mass loss of about 18.4% from the onset of heating to beforeabout 250° C., as measured by TGA.

Provided herein are methods of preparing a D-malate salt of the compoundof Formula I. In some embodiments, the method comprises slurrying acomposition comprising the compound of Formula I in a mixture ofdichloromethane and ethanol and adding a D-malic acid solution to themixture to generate the D-malate salt as a residual solid. In someembodiments, the ratio of dicholoromethane and ethanol is about 3.6:1 byvolume. In some embodiments, the ratio of dicholoromethane and ethanolis 3.3:1 by volume. In some embodiments, the D-malic acid is added in anethanol solution. In some embodiments, the solvent is used in about 46volumes. In some embodiments, the slurry is temperature cycled betweenabout 0° C. and about RT. In some embodiments, the method comprisesageing the slurry. In some embodiments, the method comprises ageing theslurry for about 13 hours. In some embodiments, the method comprisesageing the slurry for about 20 hours. In some embodiments, the methodcomprises isolating the solid through filtration.

Formula II

Provided herein is a compound of Formula II:

including pharmaceutically acceptable salts, amorphous, and polymorphforms thereof.

The compound of Formula II provided herein can be prepared using methodsknown and understood by those of ordinary skill in the art. For example,synthetic methods such as those described in U.S. Provisional App. Ser.Nos. 62/406,252; 62/447,850; 62/491,164; 62/554,817; or 62/566,093 canbe used, and these applications are herein incorporated by reference intheir entirety.

Provided herein are polymorph forms of the compound of Formula II. Theforms include, e.g., free bases, solvates, hydrates, salts, andnon-solvated forms of the compound of Formula II, including, forexample, polymorph Forms 1, 2, 7, and 8. In some embodiments, thepolymorph form of the compound of Formula II is a pharmaceuticallyacceptable salt. In some embodiments, the compound of Formula II is aphosphate salt.

Form 1

One such polymorph is a polymorph known as Form 1. Form 1 is ananhydrous polymorph of the compound of Formula II. In some embodiments,Form 1 has an X-ray powder diffraction (XRPD or XRD) pattern, obtainedwith CuKα1-radiation, with at least peaks at °2θ values of 16.5±0.2,18.9±0.2, and 26.0±0.2. In some embodiments, Form 1 has an XRPD patternwith at least peaks at °2θ values of 16.5±0.2, 18.9±0.2, 23.8±0.2,25.3±0.2, and 26.0±0.2. In some embodiments, Form 1 has an XRPD patternwith at least peaks at °2θ values of 16.5±0.2, 17.8±0.2, 18.9±0.2,23.8±0.2, 25.3±0.2, 25.6±0.2, 26.0±0.2, and 28.3±0.2. For example, insome embodiments, Form 1 has an XRPD pattern with at least peaks at °2θvalues of 9.8±0.2, 16.5±0.2, 17.8±0.2, 18.9±0.2, 23.8±0.2, 25.0±0.2,25.3±0.2, 25.6±0.2, 26.0±0.2, and 28.3±0.2.

In some embodiments, provided herein is a composition comprisingpolymorph Form 1. In some embodiments, the composition can besubstantially pure. For example, the composition has a purity of atleast about 90%. In some embodiments, the composition has a purity of atleast about 95%. In some embodiments, the composition has a purity of atleast about 98%. For example, the composition can have a purity of atleast 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%,99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%. In some embodiments, thecomposition is substantially free of other forms of the compound ofFormula II. For example, in some embodiments, the composition issubstantially free of other anhydrous forms of the compound of FormulaII. In some embodiments, the composition contains less than about 15% byweight of other forms of the compound of Formula II. For example, thecomposition can contain less than 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1% or less by weight of one or more other forms ofthe compound of Formula II. For example, the composition can containless than about 15% of Form 2, Form 7, Form 8, or a combination of twoor more thereof.

In some embodiments, provided herein is polymorph Form 1 that exhibitsan endotherm that is observed between about 185-200° C., e.g., about195° C., as measured by differential scanning calorimetry (DSC) relatedto sorbed water. In some embodiments, polymorph Form 1 exhibits anendothermic event that is observed between about 200-210° C., e.g.,about 207° C. In some embodiments, the endotherms are observed whenusing a scan rate of 10° C. per minute.

In some embodiments, provided herein is polymorph Form 1 that exhibitsan endothermic event observed from an onset of about 190° C., asmeasured by thermogravimetric/differential thermal analysis (TG/DTA). Insome embodiments, polymorph Form 1 undergoes a mass loss of about 0.4%before about 200° C., e.g., from about 190° C. to about 200° C. In someembodiments, polymorph Form 1 exhibits an endothermic event from anonset of about 204° C. In some embodiments, the endothermic event isaccompanied by a corresponding weight loss of about 0.2%.

Provided herein are methods of preparing polymorph Form 1. In someembodiments, the method comprises slurrying a composition comprising thecompound of Formula II in a solvent selected from the group consistingof 1,4-dioxane, 1-butanol, 1-propanol, acetone, anisole, chloroform,cyclohexane, cyclohexanone, dichloromethane, DMSO, ethanol, ethylacetate, isopropyl alcohol, methyl ethyl ketone, methyl acetate,2-ethoxyethanol, 2-methyl THF, methyl isobutyl ketone (MIBK),nitromethane, and THF to generate polymorph Form 1 as a residual solid.In some embodiments, the solvent is ethyl acetate. In some embodiments,the solvent is in a mixture with water, for example the solvent can be amixture of water and acetone or water and acetonitrile. In someembodiments, the water is present in an amount of about 20% by weight.In some embodiments, the water is present in an amount of about 50% byweight. In some embodiments, the slurry is temperature cycled betweenabout 40° C. and about RT. In some embodiments, the temperature cyclingoccurs between about 60 hours and about 84 hours, such as, e.g., about72 hours. In some embodiments, the method further comprises collectingthe residual solid. In some embodiments, the residual solid is collectedby filtration. In some embodiments, the method further comprises dryingthe residual solid, for example, under vacuum. In some embodiments, thedrying is at a temperature of between about 30° C. and about 50° C.,such as, e.g., about 40° C.

In some embodiments, a method of preparing a polymorph of Form 1 isprovided. The method comprises providing a composition comprising thecompound of Formula II in a solvent. In some embodiments, polymorph Form1 can be prepared by evaporating the solvent from the compositioncomprising the compound of Formula II to generate polymorph Form 1 as aresidual solid, where the solvent is selected from the group consistingof dichloromethane, DMSO, methyl acetate, 2-ethoxyethanol, nitromethane,and a mixture of acetonitrile and water (20%). In some embodiments, themethod comprises evaporating the solvent from a composition comprisingthe compound of Formula II to generate a mixture of polymorph Form 1 andanother polymorph form as a residual solid, where the solvent isselected from the group consisting of acetone, chloroform, and THF. Insome embodiments, the residual solid is a mixture of Form 1 and Form 8.

In some embodiments, polymorph Form 1 can be prepared by cooling asolution comprising the compound of Formula II in acetone to atemperature of about 5° C. to precipitate polymorph Form 1 as a residualsolid. In some embodiments, the residual solid is a mixture of Form 1and Form 8.

In some embodiments, polymorph Form 1 can be prepared by recrystallizinga composition comprising the compound of Formula II to generatepolymorph Form 1, where the recrystallizing solvent is selected from thegroup consisting of a mixture of DMSO and water and a mixture ofdichloromethane and heptane.

Form 2

Also provided herein is a polymorph known as Form 2. Form 2 is ahydrated polymorph form of the compound of Formula II. In someembodiments, Form 2 has an XRPD pattern, obtained with CuKα1-radiation,with at least peaks at °2θ values of 15.1±0.2, 17.8±0.2, and 24.2±0.2.In some embodiments, Form 2 has an XRPD pattern with at least peaks at°2θ values of 15.1±0.2, 17.8±0.2, 20.4±0.2, 21.1±0.2, and 24.2±0.2. Insome embodiments, Form 2 has an XRPD pattern with at least peaks at °2θvalues of 15.1±0.2, 17.8±0.2, 18.1±0.2, 20.4±0.2, 21.1±0.2, 23.4±0.2,24.2±0.2, and 24.6±0.2. For example, in some embodiments, Form 2 has anXRPD pattern with at least peaks at °2θ values of 6.2±0.2, 15.1±0.2,17.8±0.2, 18.1±0.2, 20.4±0.2, 21.1±0.2, 23.4±0.2, 24.2±0.2, 24.6±0.2,and 31.2±0.2.

In some embodiments, provided herein is a composition comprisingpolymorph Form 2. In some embodiments, the composition can besubstantially pure. For example, the composition has a purity of atleast about 90%. In some embodiments, the composition has a purity of atleast about 95%. In some embodiments, the composition has a purity of atleast about 98%. For example, the composition can have a purity of atleast 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%,99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%. In some embodiments, thecomposition is substantially free of other forms of the compound ofFormula II. In some embodiments, the composition contains less thanabout 15% by weight of other forms of the compound of Formula II. Forexample, the composition can contain less than 14%, 13%, 12%, 11%, 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% by weight of one or more other formsof the compound of Formula II. For example, the composition can containless than about 15% of Form 1, Form 7, Form 8, or a combination of twoor more thereof.

In some embodiments, provided herein is polymorph Form 2 that exhibitsan endotherm that is observed between about 190-200° C., e.g., about197.5° C., as measured by DSC related to sorbed water. In someembodiments, polymorph Form 2 exhibits an endothermic event that isobserved between about 200-210° C., e.g., about 207.5° C. In someembodiments, the endotherms are observed when using a scan rate of 10°C. per minute.

In some embodiments, provided herein is polymorph Form 2 that exhibits aweight loss of about 0.7% from the onset of heating to about 165° C., asmeasured by TG/DTA. In some embodiment, polymorph Form 2 exhibits anendothermic event observed from an onset of around 194° C. In someembodiments, polymorph Form 2 undergoes a mass loss of about 0.2% beforeabout 200° C., e.g., from about 194° C. to about 200° C. In someembodiments, polymorph Form 2 exhibits an endothermic event from anonset of about 205° C.

Provided herein are methods of preparing polymorph Form 2. In someembodiments, the method comprises slurrying a composition comprising thecompound of Formula II in a mixture of ethanol and water to generatepolymorph Form 2 as a residual solid. In some embodiments, the water ispresent in an amount of about 10% by weight. In some embodiments, theslurry is temperature cycled between about 40° C. and about RT. In someembodiments, the temperature cycling occurs between about 60 hours andabout 84 hours, such as, e.g., about 72 hours. In some embodiments, themethod further comprises collecting the residual solid. In someembodiments, the residual solid is collected by filtration. In someembodiments, the residual solid is dried. In some embodiments, theresidual solid is dried on the filter bed.

Form 7

Provided herein is a polymorph known as Form 7. Form 7 is a hydratedpolymorph form of the compound of Formula II. In some embodiments, Form7 has an XRPD pattern, obtained with CuKα1-radiation, with at leastpeaks at °2θ values of 16.6±0.2, 18.0±0.2, and 19.9±0.2. In someembodiments, Form 7 has an XRPD pattern with at least peaks at °2θvalues of 16.6±0.2, 18.0±0.2, 19.3±0.2, 19.9±0.2, and 23.3±0.2. In someembodiments, Form 7 has an XRPD pattern with at least peaks at °2θvalues of 16.6±0.2, 17.3±0.2, 18.0±0.2, 19.0±0.2, 19.3±0.2, 19.9±0.2,23.3±0.2, and 25.1±0.2. For example, in some embodiments, Form 7 has anXRPD pattern with at least peaks at °2θ values of 15.8±0.2, 16.6±0.2,17.3±0.2, 18.0±0.2, 19.0±0.2, 19.3±0.2, 19.91±0.2, 21.4±0.2, 23.3±0.2,and 25.1±0.2.

In some embodiments, provided herein is a composition comprisingpolymorph Form 7. In some embodiments, the composition can besubstantially pure. For example, the composition has a purity of atleast about 90%. In some embodiments, the composition has a purity of atleast about 95%. In some embodiments, the composition has a purity of atleast about 98%. For example, the composition can have a purity of atleast 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%,99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%. In some embodiments, thecomposition is substantially free of other forms of the compound ofFormula II. In some embodiments, the composition contains less thanabout 15% by weight of other forms of the compound of Formula II. Forexample, the composition can contain less than 14%, 13%, 12%, 11%, 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% by weight of one or more other formsof the compound of Formula II. For example, the composition can containless than about 15% of Form 1, Form 2, Form 8, or a combination of twoor more thereof.

In some embodiments, provided herein is polymorph Form 7 that exhibitsan endotherm that is observed between about 145-155° C., e.g., about150° C., as measured by DSC related to sorbed water. In someembodiments, polymorph Form 7 exhibits an endotherm that is observedbetween about 190-205° C., e.g., about 201° C. In some embodiments,polymorph Form 7 exhibits an endothermic event that is observed betweenabout 205-210° C., e.g., about 207° C. In some embodiments, theendotherms are observed when using a scan rate of 10° C. per minute.

In some embodiments, provided herein is polymorph Form 7 that exhibitsan endothermic event observed from an onset of about 147° C., asmeasured by TG/DTA. In some embodiments, polymorph Form 7 undergoes aweight loss of about 7% before about 150° C., e.g., from about 145° C.to about 155° C. In some embodiments, the weight loss is the loss ofsolvent. In some embodiments, the weight loss is equal to about twoequivalents of solvent as compared to the amount of compound present inthe sample. In some embodiments, the solvent is water. In someembodiments, polymorph Form 7 exhibits an endothermic event observedfrom an onset of about 196° C. In some embodiments, polymorph Form 7dehydrates upon heating to become polymorph Form 1. In some embodiments,the endothermic event relates to the transition observed in Form 1. Insome embodiments, the transition relates to the endothermic event ofForm 1 observed from an onset of about 206° C.

Provided herein are methods of preparing polymorph Form 7. In someembodiments, the method comprises slurrying a composition comprising thecompound of Formula II in a mixture of 1,4-dioxane and water to generatepolymorph Form 7 as a residual solid. In some embodiments, the water ispresent in an amount of about 10% by weight. In some embodiments, theslurry is temperature cycled between about 40° C. and about RT. In someembodiments, the temperature cycling occurs between about 60 hours andabout 84 hours, such as, e.g., about 72 hours. In some embodiments, themethod further comprises collecting the residual solid. In someembodiments, the residual solid is collected by filtration. In someembodiments, the residual solid is dried. In some embodiments, theresidual solid is dried on the filter bed.

Form 8

Provided herein is a polymorph known as Form 8. Form 8 is a solvatedpolymorph form of the compound of Formula II. Polymorph Form 8 is anisopropyl alcohol solvate polymorph form of the compound of Formula II.In some embodiments, Form 8 has an XRPD pattern, obtained withCuKα1-radiation, with at least peaks at °2θ values of 15.1±0.2,17.8±0.2, and 24.2±0.2. In some embodiments, Form 8 has an XRPD patternwith at least peaks at °2θ values of 15.1±0.2, 17.8±0.2, 20.4±0.2,21.1±0.2, and 24.2±0.2. In some embodiments, Form 8 has an XRPD patternwith at least peaks at °2θ values of 15.1±0.2, 17.8±0.2, 18.1±0.2,20.4±0.2, 21.1±0.2, 23.4±0.2, 24.2±0.2, and 24.6±0.2. For example, insome embodiments, Form 8 has an XRPD pattern with at least peaks at °2θvalues of 6.2±0.2, 15.1±0.2, 17.8±0.2, 18.1±0.2, 20.4±0.2, 21.1±0.2,23.4±0.2, 24.2±0.2, 24.6±0.2, and 31.2±0.2.

In some embodiments, provided herein is a composition comprisingpolymorph Form 8. In some embodiments, the composition can besubstantially pure. For example, the composition has a purity of atleast about 90%. In some embodiments, the composition has a purity of atleast about 95%. In some embodiments, the composition has a purity of atleast about 98%. For example, the composition can have a purity of atleast 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%,99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%. In some embodiments, thecomposition is substantially free of other forms of the compound ofFormula II. In some embodiments, the composition contains less thanabout 15% by weight of other forms of the compound of Formula II. Forexample, the composition can contain less than 14%, 13%, 12%, 11%, 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% by weight of one or more other formsof the compound of Formula II. For example, the composition can containless than about 15% of Form 1, Form 2, Form 7, or a combination of twoor more thereof.

In some embodiments, provided herein is polymorph Form 8 that exhibitsan endotherm that is observed between about 165-175° C., e.g., about172° C., as measured by DSC related to sorbed water. In someembodiments, polymorph Form 8 exhibits an endotherm that is observedbetween about 185-200° C., e.g., about 196° C. In some embodiments,polymorph Form 8 exhibits an endothermic event that is observed betweenabout 200-210° C., e.g., about 206° C. In some embodiments, theendotherms are observed when using a scan rate of 10° C. per minute.

In some embodiments, provided herein is polymorph Form 8 that exhibitsan endothermic event observed at about 165° C., as measured by TG/DTA.In some embodiments, polymorph Form 8 undergoes a weight loss of about4% before about 165° C. In some embodiments, the weight loss is the lossof solvent. In some embodiments, the weight loss is equal to about 0.5equivalents of solvent. In some embodiments, the solvent is IPA. In someembodiments, polymorph Form 8 exhibits an endothermic event observedfrom an onset of about 191° C. In some embodiments, the endothermicevent relates to the transition observed in Form 1. In some embodiments,the transition relates to the endothermic event of Form 1 observed froman onset of about 205° C.

Provided herein are methods of preparing polymorph Form 8. In someembodiments, the method comprises slurrying a composition comprising thecompound of Formula II in a solvent selected from the group consistingof IPA and 1-propanol to generate polymorph Form 8 as a residual solid.In some embodiments, the slurry is temperature cycled between about 40°C. and about RT. In some embodiments, the temperature cycling occursbetween about 60 hours and about 84 hours, such as, e.g., about 72hours. In some embodiments, the method further comprises collecting theresidual solid. In some embodiments, the residual solid is collected byfiltration. In some embodiments, the method further comprises drying theresidual solid, for example, under vacuum. In some embodiments, thedrying is at a temperature of between about 30° C. and about 50° C.,such as, e.g., about 40° C.

In some embodiments, a method of preparing a polymorph of Form 8 isprovided. The method comprises providing a composition comprising thecompound of Formula II in a solvent. In some embodiments, the methodcomprises evaporating the solvent from the composition comprising thecompound of Formula II, including amorphous and polymorph forms thereofto generate a mixture of polymorph Form 8 and another polymorph form asa residual solid. In some embodiments, the residual solid is a mixtureof polymorph Form 8 and polymorph Form 1. In some embodiments, thesolvent is acetone. In some embodiments, the solvent is chloroform. Insome embodiments, the solvent is THF.

Salts of Formula II

In some embodiments, the compound of Formula II is a pharmaceuticallyacceptable salt. For example, pharmaceutically acceptable salts of thecompound of Formula II can include, but are not limited to, sulfate,tosylate, naphthalene-2-sulfonate, oxalate, phosphate, tartrate, andfumarate salts. In some embodiments, the compound of Formula II is asulfate salt. In some embodiments, the sulfate salt is prepared in amixture of solvents. In some embodiments, the solvent is a mixture ofIPA and water. In some embodiments, the water is present in an amount ofabout 10% by weight. In some embodiments, the compound of Formula II isa tosylate salt. In some embodiments, the tosylate salt is prepared in amixture of solvents. In some embodiments, the solvent is a mixture ofacetone and water. In some embodiments, the water is present in anamount of about 10% by weight. In some embodiments, the compound ofFormula II is a naphthalene-2-sulfonate salt. In some embodiments, thenaphthalene-2-sulfonate salt is prepared in a mixture of solvents. Insome embodiments, the solvent is a mixture of THF and water. In someembodiments, the water is present in an amount of about 10% by weight.In some embodiments, the compound of Formula II is an oxalate salt. Insome embodiments, the oxalate salt is prepared in a mixture of solvents.In some embodiments, the solvent is a mixture of 1,4-dioxane and water.In some embodiments, the water is present in an amount of about 10% byweight. In some embodiments, the oxalate salt is prepared fromevaporation from a mixture of solvents. In some embodiments, the solventis a mixture of THF and water. In some embodiments, the compound ofFormula II is a tartrate salt. In some embodiments, the tartrate salt isprepared in a mixture of solvents. In some embodiments, the solvent is amixture of IPA and water. In some embodiments, the water is present inan amount of about 10% by weight. In some embodiments, the compound ofFormula II is a fumarate salt. In some embodiments, the fumarate salt isprepared in a mixture of solvents. In some embodiments, the solvent is amixture of THF and water. In some embodiments, the compound of FormulaII is a phosphate salt. In some embodiments, the phosphate salt isprepared in a mixture of solvents. In some embodiments, the solvent is amixture of acetone and water. In some embodiments, the solvent is amixture of IPA and water. In some embodiments, the water is present inan amount of about 10% by weight.

Provided herein is a phosphate salt of the compound of Formula II. Insome embodiments, the phosphate salt has a ratio of about 1.4:1,PO₄:free base. In some embodiments, the phosphate salt has an XRPDpattern, obtained with CuKα1-radiation, with at least peaks at ° 2θvalues of 3.6±0.2, 16.7±0.2, and 18.2±0.2. In some embodiments, thephosphate salt has an XRPD pattern with at least peaks at °2θ values of3.6±0.2, 15.9±0.2, 16.7±0.2, 17.8±0.2, and 18.2±0.2. In someembodiments, the phosphate salt has an XRPD pattern with at least peaksat °2θ values of 3.6±0.2, 6.2±0.2, 15.9±0.2, 16.7±0.2, 17.8±0.2,18.2±0.2, 20.3±0.2, and 25.5±0.2. For example, in some embodiments, thephosphate salt has an XRPD pattern with at least peaks at °2θ values of3.6±0.2, 6.2±0.2, 15.9±0.2, 16.7±0.2, 17.8±0.2, 18.2±0.2, 19.1±0.2,20.3±0.2, 20.9±0.2, and 25.5±0.2.

In some embodiments, provided herein is a composition comprising thephosphate salt of the compound of Formula II. In some embodiments, thecomposition can be substantially pure. For example, the composition hasa purity of at least about 90%. In some embodiments, the composition hasa purity of at least about 95%. In some embodiments, the composition hasa purity of at least about 98%. For example, the composition can have apurity of at least 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%,99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%. In some embodiments,the composition is substantially free of other forms of the compound ofFormula II. In some embodiments, the composition contains less thanabout 15% by weight of other forms of the compound of Formula II. Forexample, the composition can contain less than 14%, 13%, 12%, 11%, 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% by weight of one or more other formsof the compound of Formula II.

In some embodiments, provided herein is a phosphate salt of the compoundof Formula II that exhibits an endotherm that is observed between about165-175° C., e.g., about 170° C., as measured by DSC related to sorbedwater. In some embodiments, the endotherm is observed when using a scanrate of 10° C. per minute.

In some embodiments, provided herein is a phosphate salt of the compoundof Formula II that exhibits a melting point of about 167° C., asmeasured by TG/DTA. In some embodiments, the phosphate salt of thecompound of Formula II undergoes a mass loss of about 1.3% from theonset of heating to before about 150° C. In some embodiments, thephosphate salt of the compound of Formula II exhibits a second weightloss of about 1.2% from an onset of about 167° C.

Provided herein are methods of preparing a phosphate salt of thecompound of Formula II. In some embodiments, the method comprisesslurrying a composition comprising the compound of Formula II in amixture of water and IPA and adding a phosphoric acid solution to themixture to generate the phosphate salt as a residual solid. In someembodiments, the water is present in an amount of about 10% by weight.In some embodiments, the acid is a 1M solution of phosphoric acid. Insome embodiments, the slurry is temperature cycled between about 40° C.and about RT. In some embodiments, the temperature cycling occursbetween about 12 hours and about 48 hours, such as, e.g., about 24hours. In some embodiments, the method further comprises centrifugingthe composition and collecting the residual solid. In some embodiments,the residual solid is washed with a solvent. In some embodiments, thesolvent is IPA. In some embodiments, the method further comprises dryingthe residual solid. In some embodiments, the residual solid is driedunder vacuum. In some embodiments, the drying is at a temperature ofbetween about 30° C. and about 50° C., such as, e.g., about 40° C.

Formula III

Provided herein is a compound of Formula III:

including pharmaceutically acceptable salts, amorphous, and polymorphforms thereof.

The compound of Formula III provided herein can be prepared usingmethods known and understood by those of ordinary skill in the art. Forexample, synthetic methods such as those described in U.S. ProvisionalApp. Ser. Nos. 62/406,252; 62/447,850; 62/491,164; 62/554,817; or62/566,093 can be used, and these applications are herein incorporatedby reference in their entirety.

Provided herein are polymorph forms of the compound of Formula III. Theforms include, e.g., free bases, solvates, hydrates, salts, andnon-solvated forms of the compound of Formula III, including, forexample, polymorph Form A. In some embodiments, the polymorph form ofthe compound of Formula III is a pharmaceutically acceptable salt.

Form A

One such polymorph is a polymorph known as Form A. Form A is a polymorphform of the compound of Formula III. In some embodiments, Form A has anXRPD pattern, obtained with CuKα1-radiation, with at least peaks at °2θvalues of 17.3±0.2, 19.2±0.2, and 23.9±0.2. In some embodiments, Form Ahas an XRPD pattern with at least peaks at °2θ values of 4.7±0.2,17.3±0.2, 18.8±0.2, 19.2±0.2, and 23.9±0.2. In some embodiments, Form Ahas an XRPD pattern with at least peaks at °2θ values of 4.7±0.2,6.8±0.2, 15.2±0.2, 17.3±0.2, 18.8±0.2, 19.2±0.2, 20.2±0.2, and 23.9±0.2.For example, in some embodiments, Form A has an XRPD pattern with atleast peaks at °2θ values of 4.7±0.2, 6.8±0.2, 13.4±0.2, 15.2±0.2,15.9±0.2, 17.3±0.2, 18.8±0.2, 19.2±0.2, 20.2±0.2, and 23.9±0.2.

In some embodiments, provided herein is a composition comprisingpolymorph Form A. In some embodiments, the composition can besubstantially pure. For example, the composition has a purity of atleast about 90%. In some embodiments, the composition has a purity of atleast about 95%. In some embodiments, the composition has a purity of atleast about 98%. For example, the composition can have a purity of atleast 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%,99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%. In some embodiments, thecomposition is substantially free of other forms of the compound ofFormula III. In some embodiments, the composition contains less thanabout 15% by weight of other forms of the compound of Formula III. Forexample, the composition can contain less than 14%, 13%, 12%, 11%, 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% by weight of one or more other formsof the compound of Formula III. For example, the composition can containless than about 15% of amorphous form.

In some embodiments, provided herein is polymorph Form A that exhibitsan endotherm that is observed between about 135-150° C., e.g., about140.5° C. or 146.6° C., as measured by DSC related to sorbed water.

Provided herein are methods of preparing polymorph Form A. In someembodiments, the method comprises dissolving the compound of Formula IIIin acetonitrile and adding water to generate polymorph Form A as asolid. In some embodiments, the method comprises heating a compositioncomprising the compound of Formula III and acetonitrile to reflux. Insome embodiments, the ratio of acetonitrile and water is about 2:3 byvolume. In some embodiments, the residual solid is collected byfiltration. In some embodiments, the residual solid is dried. In someembodiments, the residual solid is dried under high vacuum. In someembodiments, the residual solid is dried at about 40-45° C. In someembodiments, the residual solid is dried overnight.

Formula IV

Provided herein is a compound of Formula IV:

including pharmaceutically acceptable salts, amorphous, and polymorphforms thereof.

The compound of Formula IV provided herein can be prepared using methodsknown and understood by those of ordinary skill in the art. For example,synthetic methods such as those described in U.S. Provisional App. Ser.Nos. 62/406,275; 62/447,849; 62/491,180; 62/531,690; or 62/566,030 canbe used, and these applications are herein incorporated by reference intheir entirety.

Provided herein are polymorph forms of the compound of Formula IV. Theforms include, e.g., free bases, solvates, hydrates, salts, andnon-solvated forms of the compound of Formula IV, including, forexample, polymorph Forms A and B. In some embodiments, the polymorphform of the compound of Formula IV is a pharmaceutically acceptablesalt.

Form A

One such polymorph is a polymorph known as Form A. Form A is a polymorphform of the compound of Formula IV. In some embodiments, Form A has anXRPD pattern, obtained with CuKα1-radiation, with at least peaks at °2θvalues of 8.3±0.2, 16.3±0.2, and 21.9±0.2. In some embodiments, Form Ahas an XRPD pattern with at least peaks at °2θ values of 8.3±0.2,16.3±0.2, 16.6±0.2, 19.4±0.2, and 21.9±0.2. In some embodiments, Form Ahas an XRPD pattern with at least peaks at °2θ values of 8.3±0.2,16.3±0.2, 16.6±0.2, 19.4±0.2, 20.0±0.2, 20.5±0.2, 21.6±0.2, and21.9±0.2. For example, in some embodiments, Form A has an XRPD patternwith at least peaks at °2θ values of 8.3±0.2, 16.3±0.2, 16.6±0.2,18.1±0.2, 18.8±0.2, 19.4±0.2, 20.0±0.2, 20.5±0.2, 21.6±0.2, and21.9±0.2.

In some embodiments, provided herein is a composition comprisingpolymorph Form A. In some embodiments, the composition can besubstantially pure. For example, the composition has a purity of atleast about 90%. In some embodiments, the composition has a purity of atleast about 95%. In some embodiments, the composition has a purity of atleast about 98%. For example, the composition can have a purity of atleast 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%,99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%. In some embodiments, thecomposition is substantially free of other forms of the compound ofFormula IV. In some embodiments, the composition contains less thanabout 15% by weight of other forms of the compound of Formula IV. Forexample, the composition can contain less than 14%, 13%, 12%, 11%, 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% by weight of one or more other formsof the compound of Formula IV. For example, the composition can containless than about 15% of Form B, amorphous form, or a combination thereof.

In some embodiments, provided herein is polymorph Form A that exhibitsan endotherm that is observed between about 145-155° C., e.g., about149.9° C., as measured by DSC related to sorbed water.

Provided herein are methods of preparing polymorph Form A. In someembodiments, the method comprises slurrying a composition comprising thecompound of Formula IV in a solvent selected from the group consistingof acetone, acetonitrile, 2-butanol, chloroform, ethanol, ethyl acetate,heptane, hexane, isopropanol, MTBE, DMSO, THF, water, and combinationsthereof to generate polymorph Form A as a residual solid. In someembodiments, the solvent is acetone, 2-butanol, or acetonitrile. In someembodiments, the solvent is in a mixture with water, for example thesolvent can be a mixture of water and acetone, water and ethanol, orwater and DMSO. In some embodiments, the water is present in an amountof about 50% by weight. In some embodiments, the water is present in anamount of about 40% by weight. In some embodiments, the solvent is in amixture with heptane, for example the solvent can be a mixture ofchloroform and heptane or heptane and acetone. In some embodiments, theheptane is present in an amount of about 50% by weight. In someembodiments, the heptane is present in an amount of about 70% by weight.In some embodiments, Form A is prepared by adding an anti-solvent into asolution of the compound of Formula IV in a solvent. In someembodiments, the anti-solvent is heptane or water. In some embodiments,the solvent is DMSO and the anti-solvent is water. In some embodiments,the vapor of an anti-solvent is diffused into a solution of the compoundof Formula IV. In some embodiments, the method further comprisescollecting the residual solid. In some embodiments, the residual solidis collected by filtration. In some embodiments, the method compriseswashing the solid. In some embodiments, the method comprises washing thesolid with water, MTBE, or a combination thereof. In some embodiments,the method further comprises drying the residual solid, for example,under vacuum.

Form B

One such polymorph is a polymorph known as Form B. Form B is a polymorphform of the compound of Formula IV. In some embodiments, Form B has anXRPD pattern, obtained with CuKα1-radiation, with at least peaks at °2θvalues of 7.5±0.2, 13.7±0.2, and 16.9±0.2. In some embodiments, Form Bhas an XRPD pattern with at least peaks at °2θ values of 7.5±0.2,9.7±0.2, 13.7±0.2, 16.9±0.2, and 19.9±0.2. In some embodiments, Form Bhas an XRPD pattern with at least peaks at °2θ values of 7.5±0.2,9.7±0.2, 13.7±0.2, 14.5±0.2, 16.9±0.2, 19.4±0.2, 19.9±0.2, and 21.3±0.2.For example, in some embodiments, Form B has an XRPD pattern with atleast peaks at °2θ values of 7.5±0.2, 9.7±0.2, 9.9±0.2, 13.7±0.2,14.5±0.2, 16.9±0.2, 19.4±0.2, 19.9±0.2, 21.3±0.2, and 27.4±0.2.

In some embodiments, provided herein is a composition comprisingpolymorph Form B. In some embodiments, the composition can besubstantially pure. For example, the composition has a purity of atleast about 90%. In some embodiments, the composition has a purity of atleast about 95%. In some embodiments, the composition has a purity of atleast about 98%. For example, the composition can have a purity of atleast 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%,99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%. In some embodiments, thecomposition is substantially free of other forms of the compound ofFormula IV. In some embodiments, the composition contains less thanabout 15% by weight of other forms of the compound of Formula IV. Forexample, the composition can contain less than 14%, 13%, 12%, 11%, 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% by weight of one or more other formsof the compound of Formula IV. For example, the composition can containless than about 15% of Form A, amorphous form, or a combination thereof.

In some embodiments, provided herein is polymorph Form B that exhibitsan endotherm that is observed between about 160-170° C., e.g., about164.6° C., as measured by DSC related to sorbed water.

Provided herein are methods of preparing polymorph Form B. In someembodiments, the method comprises slurrying a composition comprising thecompound of Formula IV in a mixture of ethanol and water to generatepolymorph Form B as a residual solid. In some embodiments, the water ispresent in an amount of about 10% by weight. In some embodiments, theslurry is aged between about 24 and about 72 hours, e.g., about 36hours. In some embodiments, the method further comprises collecting theresidual solid. In some embodiments, the residual solid is collected byfiltration. In some embodiments, the residual solid is dried. In someembodiments, the residual solid is dried in a vacuum oven. In someembodiments, the residual solid is dried with nitrogen bleed. In someembodiments, the residual solid is dried at room temperature. In someembodiments, the residual solid is dried between about 10 and about 20hours, e.g., about 18 hours.

It will be understood that the 2-theta values of the XRPD patterns forthe crystalline forms of the compound of Formula I-IV, e.g., Forms A ofFormula I, Forms 1, 2, 7, and 8 of Formula II, Form A of Formula III, orForms A and B of Formula IV, and pharmaceutically acceptable saltsthereof, e.g., chloride salt, bromide salt, malate salt, and phosphatesalt, can vary slightly from one instrument to another and alsodepending on variations in sample preparation and batch to batchvariation, and so the values quoted are not to be construed as absolute.It will be understood that the peak positions in an XRPD pattern arereported in terms of angular positions (two theta) with an allowablevariability of ±0.2° 2θ. The variability of ±0.2° 2θ is intended to beused when comparing two powder XRPD patterns. In practice, if adiffraction pattern peak from one pattern is assigned a range of angularpositions (two theta) which is the measured peak position ±0.2° and ifthose ranges of peak positions overlap, then the two peaks areconsidered to have the same angular position. For example, if a peakfrom one pattern is determined to have a position of 11.0° 2θ, forcomparison purposes the allowable variability allows the peak to beassigned a position in the range of 10.8°-11.2° 2θ. It will also beunderstood that the relative intensities of peaks can vary depending onorientation effects so that the intensities shown in the XRPD tracesincluded herein are illustrative and not intended to be used forabsolute comparison. It is to be further understood that for comparisonpurposes some variability in peak intensities from those shown in XRPDtraces is allowed. Accordingly, it is to be understood that the phrase“substantially the same XRPD pattern as shown in FIG. 1” means that forcomparison purposes, at least 90% of the peaks shown in FIG. 1 arepresent.

Compounds provided herein can also contain unnatural proportions ofatomic isotopes at one or more of the atoms that constitute suchcompounds. That is, an atom, in particular when mentioned in relation toa compound according to Formula I-IV, comprises all isotopes andisotopic mixtures of that atom, such as naturally occurring isotopeswith natural abundance. For example, when hydrogen is mentioned, it isunderstood to refer to ¹H, ²H, ³H or mixtures thereof; when carbon ismentioned, it is understood to refer to ¹²C, ¹³C, ¹⁴C or mixturesthereof; when nitrogen is mentioned, it is understood to refer to ¹⁴N,¹⁵N or mixtures thereof; and when oxygen is mentioned, it is understoodto refer to ¹⁶O, ¹⁷O, ¹⁸O or mixtures thereof. All isotopic variationsof the compounds provided herein are intended to be encompassed withinthe scope of the present invention.

For illustrative purposes, Schemes 1-6 show general methods forpreparing the compounds provided herein as well as key intermediates.For a more detailed description of the individual reaction steps, see,e.g., U.S. Provisional App. Ser. Nos. 62/329,895, 62/406,252,62/447,850, 62/566,093, and 62/566,030 all of which are incorporated byreference in their entirety herein. Those skilled in the art willappreciate that other synthetic routes can be used to synthesize thecompounds. Although specific starting materials and reagents aredepicted in the Schemes and discussed below, other starting materialsand reagents can be easily substituted to provide a variety ofderivatives and/or reaction conditions.

Scheme 1 shows a general scheme for the synthesis of the compound ofFormula I (shown as compound 13 and 13a for Formula I in scheme 1),where B is 1-methyl-1H-pyrazole-4-yl; X¹ is N; X², X³, and X⁴ are CH;and D and E are represented by

where the wavy line indicates the point of attachment to the ringcomprising X¹, X², X³, and X⁴.

Compound 2 is obtained by treating MSH reagent with3-bromo-5-methoxypyridine, which is commercially available. Theaminating reagent O-mesitylsulfonylhydroxylamine (MSH) may be preparedas described in Mendiola, J., et al., Org. Process Res. Dev. 2009,13(2), 263-267. Compound 2 may be reacted with ethyl propiolate toprovide the pyrazolo[1,5-a]pyridine a mixture of compounds 3A and 3B,which typically are obtained in a ratio of approximately 2:1 to 9:1. Themixture of compounds 3A and 3B may be treated with 48% HBr at elevatedtemperatures, followed by recrystallization or chromatographypurifications to isolate compound 4A as the minor isomer and compound 4Bas the major isomer.

The isolated compound 4B may be functionalized with a formyl group usingPOCl₃ followed by purification to provide compound 5. The formyl groupof compound 5 may be converted to an oxime group using NH₂OH to providecompound 6. The oxime group of compound 6 may be converted to a nitrilegroup using acetic anhydride to provide compound 7. The B group may beinstalled by treating compound 7 with a corresponding boronic esterhaving the formula hetAr¹—B(OR^(a))(OR^(b)) where hetAr¹ is1-methyl-1H-pyrazole-4-yl as defined in Formula I, using appropriatepalladium-catalyzed cross-coupling reaction conditions, e.g., Suzukicoupling reaction conditions (for example, a palladium catalyst andoptionally a ligand in the presence of an inorganic base, for example,Pd₂(dba)₃, X-Phos and Na₂CO₃ in dioxane at elevated temperatures) toprovide compound 8 where B is 1-methyl-1H-pyrazole-4-yl as defined inFormula I. The methoxy group of compound 8 may be converted to a hydroxygroup by treating compound 8 with aluminum trichloride to providecompound 9. The free hydroxy group of compound 9 may be converted to atriflate group by treating compound 9 with a triflating reagent, forexample1,1,1-trifluoro-N-phenyl-N-((trifluoromethyl)sulfonyl)methanesulfonamideto provide compound 10. Compound 12 may be prepared by coupling compound10 with the corresponding boronic ester compound 11 where Ring D is

wherein the wavy line indicates the point of attachment of Ring D to thering comprising X¹, X², X³ and X⁴, and the asterisk indicates the pointof attachment to P¹; X¹, X², X³ and X⁴ are as defined above; P¹ is anamino protecting group; Z is —B(OR^(x))(OR^(y)) Z is —B(OR^(a))(OR^(b))and R^(a) and R^(b) are H or C1-C6 alkyl, or R^(a) and R^(b) togetherwith the atoms to which they are connected form a 5-6 membered ringoptionally substituted with one to four C1-C3 alkyl groups, usingappropriate palladium-catalyzed cross-coupling reaction conditions,e.g., Suzuki coupling reaction conditions (for example, a palladiumcatalyst and optionally a ligand in the presence of an inorganic base,for example, Pd₂(dba)₃, X-Phos and Na₂CO₃ in dioxane at elevatedtemperatures). The protecting group on the D ring of compound 12 may beremoved under standard conditions (for example, a Boc protecting groupmay be removed by treating compound 12 under acidic conditions, e.g.,using HCl). The deprotected D ring may be functionalized (i.e., reactedor treated with an appropriate reagent) to introduce the E group understandard conditions such as described below to provide compound 13 whereE is

Alternatively, compound 10 may be coupled with compound 14 usingappropriate palladium-catalyzed cross-coupling reaction conditions,e.g., Suzuki coupling reaction conditions (for example, a palladiumcatalyst and optionally a ligand in the presence of an inorganic base,for example, Pd(PPh₃)₄ and Na₂CO₃) to provide compound 15. Compound 15may be reacted with compound 16 under appropriate SNAr conditions (forexample, optionally in the presence of a base such as K₂CO₃ and atelevated temperature) to provide compound 12a, wherein the D ring ofcompound 16 is

wherein the wavy line indicates the point of attachment of Ring D to thering comprising X¹, X², X³ and X⁴, and the asterisk indicates the pointof attachment to P¹; X³ and X⁴ are as defined above; P¹ is an aminoprotecting group; Z is —B(OR^(x))(OR^(y)), the second nitrogen atom isprotected with an appropriate amine protecting group prior to coupling.The protecting group if present on the D ring of compound 12a may beremoved under standard conditions (for example, a Boc group may beremoved by treating compound 12a to acidic conditions, e.g., HCl). Thedeprotected D ring may be functionalized (i.e., reacted or treated withan appropriate reagent) to introduce the E group under standardconditions such as described below to provide compound 13a where E is

Scheme 2 shows an alternative route for the synthesis of compound 13,wherein B, X¹, X², X³, X⁴, D and E are as defined in Scheme 1. Compound4A (prepared as in Scheme 1) may be functionalized with a formyl groupusing POCl₃ to provide compound 17. The formyl group may be converted toan oxime group using NH₂OH to provide compound 18. The oxime group maybe converted to a nitrile group using acetic anhydride to providecompound 19. The methoxy group of compound 19 may be converted to ahydroxy group by treating compound 19 with aluminum trichloride toprovide compound 20. Compound 21 may be prepared by coupling compound 20with the corresponding boronic ester compound 11 where Ring D is

wherein the wavy line indicates the point of attachment of Ring D to thering comprising X¹, X², X³ and X⁴, and the asterisk indicates the pointof attachment to P¹; X¹, X², X³ and X⁴ are as defined above; P¹ is anamino protecting group; Z is —B(OR^(a))(OR^(b)) and R^(a) and R^(b) areH or C1-C6 alkyl, or R^(a) and R^(b) together with the atoms to whichthey are connected form a 5-6 membered ring optionally substituted withone to four C1-C3 alkyl groups, using appropriate palladium-catalyzedcross-coupling reaction conditions, e.g., Suzuki coupling reactionconditions (for example, a palladium catalyst and optionally a ligand inthe presence of an inorganic base, for example, Pd(PPh₃)₄ and Na₂CO₃ indioxane at elevated temperatures). The unsubstituted nitrogen atom ofthe D ring is protected with an appropriate amine protecting group priorto coupling. The free hydroxy group of compound 21 may be converted to atriflate group by treating compound 21 with a triflating reagent, forexample1,1,1-trifluoro-N-phenyl-N-((trifluoromethyl)sulfonyl)methanesulfonamideto provide compound 22. The B group may be installed by treatingcompound 22 with the corresponding boronic ester having the formulahetAr¹—B(OR^(a))(OR^(b)) where hetAr¹ is 1-methyl-1H-pyrazole-4-yl asdefined in Formula I and R^(a) and R^(b) are H or C1-C6 alkyl, or R^(a)and R^(b) together with the atoms to which they are connected form a 5-6membered ring optionally substituted with one to four C1-C3 alkylgroups, using appropriate palladium-catalyzed cross-coupling reactionconditions, e.g., Suzuki coupling reaction conditions (for example, apalladium catalyst and optionally a ligand in the presence of aninorganic base, for example, Pd₂(dba)₃, X-Phos and Na₂CO₃ in dioxane atelevated temperatures) to provide compound 12 where B is1-methyl-1H-pyrazole-4-yl as defined in Formula I. The protecting groupif present on the D ring of compound 12 may be removed under standardconditions (for example, a Boc group may be removed by treating compound12 to acidic conditions, e.g., HCl in propan-2-ol). The deprotected Dring may be functionalized (i.e., reacted or treated with an appropriatereagent) to introduce the E group under standard conditions such asdescribed below to provide compound 13 where E is

Scheme 3 shows a general scheme for the synthesis of the compound ofFormula II or Formula III (shown as compound 12 for Formula II or III inscheme 3), where B is —CH₂C(CH₃)₂OH; X¹ is N; X², X³, and X⁴ are CH; andD and E are represented by

respectively, where the wavy line indicates the point of attachment tothe ring comprising X¹, X², X³, and X⁴.

Compound 2 is obtained by treating commercially available3-bromo-5-methoxypyridine (compound 1) withO-(mesitylsulfonyl)hydroxylamine. The O-mesitylsulfonylhydroxylamine canbe prepared as described in Mendiola et al., Org. Process Res. Dev.(2009) 13(2):263-267. Compound 2 can be reacted with ethyl propiolate toprovide a mixture of compounds 3A and 3B, which typically are obtainedin a ratio of approximately 2:1 to 9:1, respectively. The mixture ofcompounds 3A and 3B can be treated with 48% HBr at elevatedtemperatures, followed by recrystallization or chromatographypurifications, to isolate compound 4A as the minor isomer and compound4B as the major isomer. After isolation, compound 4A can be treated withPOCl₃ to provide compound 5. The formyl group can be converted to anoxime group using NH₂OH to provide compound 6. The oxime group can beconverted to a nitrile group using acetic anhydride to provide compound7. The methoxy group of compound 7 can be converted to a hydroxy groupby treating compound 7 with aluminum trichloride to provide compound 8.

To prepare compound 9, compound 8 can be reacted with a reagent such as

where X is a leaving atom or group (such as a halide or triflate), inthe presence of a suitable base (e.g., a metal alkali carbonate, such aspotassium carbonate). Compound 11 can then be prepared by couplingcompound 9 with the corresponding boronic ester compound 10 (where RingD is

wherein the wavy line indicates the point of attachment of Ring D to thering comprising X¹, X², X³ and X⁴, and the asterisk indicates the pointof attachment to P¹; X¹, X², X³ and X⁴ are as defined above; P¹ is anamino protecting group; Z is —B(OR^(x))(OR^(y)) and R^(z) and R^(y) areH or (1-6C)alkyl, or R^(x) and R^(y) together with the atoms to whichthey are connected form a 5-6 membered ring optionally substituted with1-4 substituents selected from (C1-C3 alkyl)) using appropriatepalladium-catalyzed cross-coupling reaction conditions, e.g., Suzukicoupling reaction conditions (for example, a palladium catalyst andoptionally a ligand in the presence of an inorganic base, for example,Pd(PPh₃)₄ and Na₂CO₃ in dioxane at elevated temperatures). Compound 12can then be prepared from compound 11 by removing the protecting groupP¹ under standard conditions (for example, a Boc group can be removed bytreating compound 11 under acidic conditions, e.g., HCl), followed byfunctionalization (i.e., reacting or treating compound 11 with theappropriate reagent) to introduce the E group

under standard conditions.

Alternatively, compound 8 can be coupled with the corresponding boronicester compound 10 to provide compound 11a using appropriatepalladium-catalyzed cross-coupling reaction conditions, e.g., Suzukicoupling reaction conditions (for example, a palladium catalyst andoptionally a ligand in the presence of an inorganic base, for example,Pd(PPh₃)₄ and Na₂CO₃ in dioxane at elevated temperatures). Compound 11acan then be reacted with a reagent such as

where X is a leaving atom or group (such as a halide or triflate), underMitsunobu reaction conditions (e.g., PPh₃ and diisopropylazodicarboxylate) to provide compound 11. Compound 12 can then beprepared from compound 11 as described above.

Scheme 4 shows another general scheme for the synthesis of compound 12where B,

X³, X⁴, Ring D, and E are as defined above for Scheme 3.

Compound 9 (prepared, e.g., as described in Scheme 3) in which B is asdefined above, can be coupled with the corresponding boronic ester 13(where X¹, X², X³ and X⁴ are as defined above; L² is a leaving groupsuch as a triflate or halide); Z is —B(OR^(x))(OR^(y)) and R^(z) andR^(y) are H or (1-6C)alkyl, or R^(x) and R^(y) together with the atomsto which they are connected form a 5-6 membered ring optionallysubstituted with 1-4 substituents selected from (C1-C3 alkyl)), usingappropriate palladium-catalyzed cross-coupling reaction conditions,e.g., Suzuki coupling reaction conditions (for example, a palladiumcatalyst and optionally a ligand in the presence of an inorganic base,for example, Pd(PPh₃)₄ and Na₂CO₃ in dioxane at elevated temperatures)to provide compound 14. Compound 16 can be prepared by coupling compound14 with compound 15 where Ring D is as defined above and P¹ is an aminoprotecting group, under appropriate SNAr conditions (for example,optionally in the presence of a base such as K₂CO₃ and at elevatedtemperature).

The protecting group P¹ on Ring D of compound 16 can be removed understandard conditions (for example, a Boc group can be removed by treatingcompound 16 under acidic conditions, e.g., HCl) to provide compound 12where E is H (i.e., Ring D is deprotected). The deprotected Ring D canthen be functionalized (i.e., reacted or treated with an appropriatereagent) to introduce the E group under standard conditions such asdescribed below to provide compound 12 where E is as defined above forScheme 3.

Scheme 5 shows a general scheme for the synthesis of the compound ofFormula IV (shown as compound 12 for Formula IV in scheme 5), where B is—CH₂C(CH₃)₂OH; X¹ is N; X², X³, and X⁴ are CH; and D, E, (R^(a))_(n),and (R^(b))_(m) are represented by

where the wavy line indicates the point of attachment to the ringcomprising X¹, X², X³, and X⁴.

Compound 2 is obtained by treating 3-bromo-5-methoxypyridine (compound1), which is commercially available, withO-(mesitylsulfonyl)hydroxylamine. The O-mesitylsulfonylhydroxylamine maybe prepared as described in Mendiola, J., et al., Org. Process Res. Dev.2009, 13(2), 263-267. Compound 2 may be reacted with ethyl propiolate toprovide a mixture of compounds 3A and 3B, which typically are obtainedin a ratio of approximately 2:1 to 9:1, respectively. The mixture ofcompounds 3A and 3B may be treated with 48% HBr at elevatedtemperatures, followed by recrystallization or chromatographypurifications, to isolate compound 4A as the minor isomer and compound4B as the major isomer. After isolation, compound 4A may be treated withPOCl₃ to provide compound 5. The formyl group may be converted to anoxime group using NH₂OH to provide compound 6. The oxime group may beconverted to a nitrile group using acetic anhydride to provide compound7. The methoxy group of compound 7 may be converted to a hydroxy groupby treating compound 7 with aluminum trichloride to provide compound 8.

Compound 11a may be reacted with a reagent such a reagent such as

where X is a leaving atom or group (such as a halide or triflate), underMitsunobu reaction conditions (PPh₃ and diisopropyl azodicarboxylate) toprovide compound 11. Compound 12 may then be prepared from compound 11as described above.

Alternatively, compound 9 may be prepared by reacting compound 8 with areagent such as

and X is a leaving atom or group (such as a halide or triflate), in thepresence of a base (for example, an alkali metal carbonate, such aspotassium carbonate). Compound 11 may then be prepared by couplingcompound 9 with the corresponding boronic ester compound 10 usingappropriate palladium-catalyzed cross-coupling reaction conditions,e.g., Suzuki coupling reaction conditions (for example, a palladiumcatalyst and optionally a ligand in the presence of an inorganic base,for example, Pd(PPh₃)₄ and Na₂CO₃ in dioxane at elevated temperatures).

Scheme 6 shows another general scheme for the synthesis of compound 12where B, X¹, X², X³, X⁴, Ring D and E are as defined above for Scheme 5.

Compound 9 (prepared, e.g., as described in Scheme 5) in which B is asdefined for Scheme 5, may be coupled with compound 13 (where X¹, X², X³and X⁴ are as defined for Scheme 5; L² is a leaving group such as atriflate or halide); Z is —B(OR^(x))(OR^(y)) and R^(z) and R^(y) are Hor (1-6C) alkyl, or R^(x) and R^(y) together with the atoms to whichthey are connected form a 5-6 membered ring optionally substituted with1-4 substituents selected from (C1-C3 alkyl)), using appropriatepalladium-catalyzed cross-coupling reaction conditions, e.g., Suzukicoupling reaction conditions (for example, a palladium catalyst andoptionally a ligand in the presence of an inorganic base, for example,Pd(PPh₃)₄ and Na₂CO₃ in dioxane at elevated temperatures) to providecompound 14. Compound 12 may be prepared by coupling compound 14 withcompound 15 under appropriate SNAr conditions (for example, optionallyin the presence of a base such as K₂CO₃ and at elevated temperature)where compound 15 is defined as

or salts thereof as described in Formula IV.

“amino protecting group” as used herein refers to a derivative of thegroups commonly employed to block or protect an amino group whilereactions are carried out on other functional groups on the compound.Examples of suitable protecting groups for use in any of the processesdescribed herein include carbamates, amides, alkyl and aryl groups,imines, as well as many N-heteroatom derivatives which can be removed toregenerate the desired amine group. Non-limiting examples of aminoprotecting groups are acetyl, trifluoroacetyl, t-butyloxycarbonyl(“Boc”), benzyloxycarbonyl (“CBz”) and 9-fluorenylmethyleneoxycarbonyl(“Fmoc”). Further examples of these groups, and other protecting groups,are found in T. W. Greene et al., Greene's Protective Groups in OrganicSynthesis. New York: Wiley Interscience, 2006.

Hydroxy groups can be protected with any convenient hydroxy protectinggroup, for example as described in T. W. Greene et al., Greene'sProtective Groups in Organic Synthesis. New York: Wiley Interscience,2006. Examples include benzyl, trityl, silyl ethers, and the like.

Nitrogen atoms in compounds described in any of the above methods can beprotected with any convenient nitrogen protecting group, for example asdescribed in Greene & Wuts, eds., “Protecting Groups in OrganicSynthesis,” 2^(nd) ed. New York; John Wiley & Sons, Inc., 1991. Examplesof nitrogen protecting groups include acyl and alkoxycarbonyl groups,such as t-butoxycarbonyl (BOC), phenoxycarbonyl, and[2-(trimethylsilyl)ethoxy]methyl (SEM).

3. Methods of Treatment

The ability of the compound of Formula I-IV, including polymorph formsand pharmaceutically acceptable salts thereof, to act as a RET inhibitorcan be demonstrated by the assays described in Examples 8 and 9.

In some embodiments, the compounds provided herein exhibit potent andselective RET inhibition. For example, the compounds provided hereinexhibit nanomolar potency against wild type RET and a RET kinase encodedby a RET gene including an activating mutation or a RET kinase inhibitorresistance mutation, including, for example, the KIF5B-RET fusion, G810Rand G810S ATP cleft front mutations, M918T activating mutation, andV804M, V804L, and V804E gatekeeper mutations, with minimal activityagainst related kinases.

In some embodiments, the compounds provided herein exhibit nanomolarpotency against an altered RET fusion protein encoded by a RET geneencoding the RET fusion protein (e.g. any of the RET fusion proteinsdescribed herein including, without limitation, CCDC6-RET or KIF5B-RET)which RET gene includes a RET kinase inhibitor resistance mutation(e.g., any of the RET mutations described herein including, withoutlimitation, V804M, V804L, or V804E) such that the altered RET protein isa RET fusion protein that exhibits RET kinase resistance due to thepresence of a RET kinase inhibitor resistance amino acid substitution ordeletion. Non-limiting examples include CCDC6-RET-V804M andKIF5B-RET-V804M. In some embodiments, the compounds provided hereinexhibit nanomolar potency against an altered RET protein encoded by aRET gene that that includes a RET mutation (e.g. any of the RETmutations described herein including, without limitation, C634W orM918T) and that includes a RET kinase inhibitor resistance mutation(e.g., any of the RET kinase inhibitor resistance mutations describedherein including, without limitation, V804M, V804L, or V804E) such thatthe altered RET protein includes a RET substitution caused by the RETmutation (e.g., a RET primary mutation) and the altered RET proteinexhibits RET kinase resistance due to the presence of a RET kinaseinhibitor resistance amino acid substitution or deletion.

In some embodiments, the compounds of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,selectively target a RET kinase. For example, a compound of FormulaI-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphform thereof, can selectively target a RET kinase over another kinase ornon-kinase target.

In some embodiments, a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof, exhibits at leasta 30-fold selectivity for a RET kinase over another kinase. For example,a compound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, exhibits at least a 40-foldselectivity; at least a 50-fold selectivity; at least a 60-foldselectivity; at least a 70-fold selectivity; at least a 80-foldselectivity; at least a 90-fold selectivity; at least 100-foldselectivity; at least 200-fold selectivity; at least 300-foldselectivity; at least 400-fold selectivity; at least 500-foldselectivity; at least 600-fold selectivity; at least 700-foldselectivity; at least 800-fold selectivity; at least 900-foldselectivity; or at least 1000-fold selectivity for a RET kinase overanother kinase. In some embodiments, selectivity for a RET kinase overanother kinase is measured in a cellular assay (e.g., a cellular assayas provided herein).

In some embodiments, the compounds provided herein can exhibitselectivity for a RET kinase over a KDR kinase (e.g., VEGFR2). In someembodiments, the selectivity for a RET kinase over a KDR kinase isobserved without loss of potency for a RET kinase encoded by a RET geneincluding an activating mutation or a RET kinase inhibitor resistancemutation (e.g., a gatekeeper mutant). In some embodiments, theselectivity over a KDR kinase is at least 10-fold (e.g., at least a40-fold selectivity; at least a 50-fold selectivity; at least a 60-foldselectivity; at least a 70-fold selectivity; at least a 80-foldselectivity; at least a 90-fold selectivity; at least 100-foldselectivity; at least 150-fold selectivity; at least 200-foldselectivity; at least 250-fold selectivity; at least 300-foldselectivity; at least 350-fold selectivity; or at least 400-foldselectivity) as compared to the inhibition of KIF5B-RET (e.g., thecompounds are more potent against KIF5B-RET than KDR). In someembodiments, the selectivity for a RET kinase over a KDR kinase is about30-fold. In some embodiments, the selectivity for a RET kinase over aKDR kinase is at least 100-fold. In some embodiments, the selectivityfor a RET kinase over a KDR kinase is at least 150-fold. In someembodiments, the selectivity for a RET kinase over a KDR kinase is atleast 400-fold. Without being bound by any theory, potent KDR kinaseinhibition is believed to be a common feature among multikinaseinhibitors (MKIs) that target RET and may be the source of thedose-limiting toxicities observed with such compounds.

In some embodiments, inhibition of V804M is similar to that observed forwild-type RET. For example, inhibition of V804M is within about 2-fold(e.g., about 5-fold, about 7-fold, about 10-fold) of inhibition ofwild-type RET (e.g., the compounds were similarly potent againstwild-type RET and V804M). In some embodiments, selectivity for awildtype or V804M RET kinase over another kinase is measured in anenzyme assay (e.g., an enzyme assay as provided herein). In someembodiments, the compounds provided herein exhibit selectivecytotoxicity to RET-mutant cells.

In some embodiments, inhibition of G810S and/or G810R is similar to thatobserved for wild-type RET. For example, inhibition of G810S and/orG810R is within about 2-fold (e.g., about 5-fold, about 7-fold, about10-fold) of inhibition of wild-type RET (e.g., the compounds weresimilarly potent against wild-type RET and G810S and/or G810R). In someembodiments, selectivity for a wildtype or G810S and/or G810R RET kinaseover another kinase is measured in an enzyme assay (e.g., an enzymeassay as provided herein). In some embodiments, the compounds providedherein exhibit selective cytotoxicity to RET-mutant cells.

In some embodiments, the compounds provided herein exhibit brain and/orcentral nervous system (CNS) penetrance. Such compounds are capable ofcrossing the blood brain barrier and inhibiting a RET kinase in thebrain and/or other CNS structures. In some embodiments, the compoundsprovided herein are capable of crossing the blood brain barrier in atherapeutically effective amount. For example, treatment of a patientwith cancer (e.g., a RET-associated cancer such as a RET-associatedbrain or CNS cancer) can include administration (e.g., oraladministration) of the compound to the patient. In some suchembodiments, the compounds provided herein are useful for treating aprimary brain tumor or metastatic brain tumor. For example, aRET-associated primary brain tumor or metastatic brain tumor.

In some embodiments, the compounds of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,exhibit one or more of high GI absorption, low clearance, and lowpotential for drug-drug interactions.

Compounds of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof are useful for treating diseasesand disorders which can be treated with a RET kinase inhibitor, such asRET-associated diseases and disorders, e.g., proliferative disorderssuch as cancers, including hematological cancers and solid tumors (e.g.,advanced solid tumors and/or RET-fusion positive solid tumors), andgastrointestinal disorders such as IBS.

In certain embodiments, compounds of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof are useful forpreventing diseases and disorders as defined herein (for example,autoimmune diseases, inflammatory diseases, and cancer). The term“preventing” as used herein means the prevention of the onset,recurrence or spread, in whole or in part, of the disease or conditionas described herein, or a symptom thereof.

The term “RET-associated disease or disorder” as used herein refers todiseases or disorders associated with or having a dysregulation of a RETgene, a RET kinase (also called herein RET kinase protein), or theexpression or activity or level of any (e.g., one or more) of the same(e.g., any of the types of dysregulation of a RET gene, a RET kinase, aRET kinase domain, or the expression or activity or level of any of thesame described herein). Non-limiting examples of a RET-associateddisease or disorder include, for example, cancer and gastrointestinaldisorders such as irritable bowel syndrome (IBS).

The term “RET-associated cancer” as used herein refers to cancersassociated with or having a dysregulation of a RET gene, a RET kinase(also called herein RET kinase protein), or expression or activity, orlevel of any of the same. Non-limiting examples of a RET-associatedcancer are described herein.

The phrase “dysregulation of a RET gene, a RET kinase, or the expressionor activity or level of any of the same” refers to a genetic mutation(e.g., a chromosomal translocation that results in the expression of afusion protein including a RET kinase domain and a fusion partner, amutation in a RET gene that results in the expression of a RET proteinthat includes a deletion of at least one amino acid as compared to awildtype RET protein, a mutation in a RET gene that results in theexpression of a RET protein with one or more point mutations as comparedto a wildtype RET protein, a mutation in a RET gene that results in theexpression of a RET protein with at least one inserted amino acid ascompared to a wildtype RET protein, a gene duplication that results inan increased level of RET protein in a cell, or a mutation in aregulatory sequence (e.g., a promoter and/or enhancer) that results inan increased level of RET protein in a cell), an alternative splicedversion of a RET mRNA that results in a RET protein having a deletion ofat least one amino acid in the RET protein as compared to the wild-typeRET protein), or increased expression (e.g., increased levels) of awildtype RET kinase in a mammalian cell due to aberrant cell signalingand/or dysregulated autocrine/paracrine signaling (e.g., as compared toa control non-cancerous cell). As another example, a dysregulation of aRET gene, a RET protein, or expression or activity, or level of any ofthe same, can be a mutation in a RET gene that encodes a RET proteinthat is constitutively active or has increased activity as compared to aprotein encoded by a RET gene that does not include the mutation. Forexample, a dysregulation of a RET gene, a RET protein, or expression oractivity, or level of any of the same, can be the result of a gene orchromosome translocation which results in the expression of a fusionprotein that contains a first portion of RET that includes a functionalkinase domain, and a second portion of a partner protein (i.e., that isnot RET). In some examples, dysregulation of a RET gene, a RET protein,or expression or activity or level of any of the same can be a result ofa gene translocation of one RET gene with another non-RET gene.Non-limiting examples of fusion proteins are described in Table 1.Non-limiting examples of RET kinase protein pointmutations/insertions/deletions are described in Tables 2 and 2a.Additional examples of RET kinase protein mutations (e.g., pointmutations) are RET inhibitor resistance mutations. Non-limiting examplesof RET inhibitor resistance mutations are described in Tables 3 and 4.

In some embodiments, dysregulation of a RET gene, a RET kinase, or theexpression or activity or level of any of the same can be caused by anactivating mutation in a RET gene (see, e.g., chromosome translocationsthat result in the expression of any of the fusion proteins listed inTable 1). In some embodiments, dysregulation of a RET gene, a RETkinase, or the expression or activity or level of any of the same can becaused by a genetic mutation that results in the expression of a RETkinase that has increased resistance to inhibition by a RET kinaseinhibitor and/or a multi-kinase inhibitor (MKI), e.g., as compared to awildtype RET kinase (see, e.g., the amino acid substitutions in Tables 3and 4). In some embodiments, dysregulation of a RET gene, a RET kinase,or the expression or activity or level of any of the same can be causedby a mutation in a nucleic acid encoding an altered RET protein (e.g., aRET fusion protein or a RET protein having a mutation (e.g., a primarymutation)) that results in the expression of an altered RET protein thathas increased resistance to inhibition by a RET kinase inhibitor and/ora multi-kinase inhibitor (MKI), e.g., as compared to a wildtype RETkinase (see, e.g., the amino acid substitutions in Tables 3 and 4). Theexemplary RET kinase point mutations, insertions, and deletions shown inTables 2 and 2a can be caused by an activating mutation and/or canresult in the expression of a RET kinase that has increased resistanceto inhibition by a RET kinase inhibitor and/or a multi-kinase inhibitor(MKI).

The term “activating mutation” describes a mutation in a RET kinase genethat results in the expression of a RET kinase that has an increasedkinase activity, e.g., as compared to a wildtype RET kinase, e.g., whenassayed under identical conditions. For example, an activating mutationcan result in the expression of a fusion protein that includes a RETkinase domain and a fusion partner. In another example, an activatingmutation can be a mutation in a RET kinase gene that results in theexpression of a RET kinase that has one or more (e.g., two, three, four,five, six, seven, eight, nine, or ten) amino acid substitutions (e.g.,any combination of any of the amino acid substitutions described herein)that has increased kinase activity, e.g., as compared to a wildtype RETkinase, e.g., when assayed under identical conditions. In anotherexample, an activating mutation can be a mutation in a RET kinase genethat results in the expression of a RET kinase that has one or more(e.g., two, three, four, five, six, seven, eight, nine, or ten) aminoacids deleted, e.g., as compared to a wildtype RET kinase, e.g., whenassayed under identical conditions. In another example, an activatingmutation can be a mutation in a RET kinase gene that results in theexpression of a RET kinase that has at least one (e.g., at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 12, at least 14, at least 16, at least18, or at least 20) amino acid inserted as compared to a wildtype RETkinase, e.g., the exemplary wildtype RET kinase described herein, e.g.,when assayed under identical conditions. Additional examples ofactivating mutations are known in the art.

The term “wildtype” or “wild-type” describes a nucleic acid (e.g., a RETgene or a RET mRNA) or protein (e.g., a RET protein) that is found in asubject that does not have a RET-associated disease, e.g., aRET-associated cancer (and optionally also does not have an increasedrisk of developing a RET-associated disease and/or is not suspected ofhaving a RET-associated disease), or is found in a cell or tissue from asubject that does not have a RET-associated disease, e.g., aRET-associated cancer (and optionally also does not have an increasedrisk of developing a RET-associated disease and/or is not suspected ofhaving a RET-associated disease).

The term “regulatory agency” refers to a country's agency for theapproval of the medical use of pharmaceutical agents with the country.For example, a non-limiting example of a regulatory agency is the U.S.Food and Drug Administration (FDA).

Provided herein is a method of treating cancer (e.g., a RET-associatedcancer) in a patient in need of such treatment, the method comprisingadministering to the patient a therapeutically effective amount of acompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof or a pharmaceutical compositionthereof. For example, provided herein are methods for treating aRET-associated cancer in a patient in need of such treatment, the methodcomprising a) detecting a dysregulation of a RET gene, a RET kinase, orthe expression or activity or level of any of the same in a sample fromthe patient; and b) administering a therapeutically effective amount ofa compound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof. In some embodiments, thedysregulation of a RET gene, a RET kinase, or the expression or activityor level of any of the same includes one or more fusion proteins.Non-limiting examples of RET gene fusion proteins are described inTable 1. In some embodiments, the fusion protein is KIF5B-RET. In someembodiments, the dysregulation of a RET gene, a RET kinase, or theexpression or activity or level of any of the same includes one or moreRET kinase protein point mutations/insertions. Non-limiting examples ofRET kinase protein point mutations/insertions/deletions are described inTables 2 and 2a. In some embodiments, the RET kinase protein pointmutations/insertions/deletions are selected from the group consisting ofM918T, M918V, C634W, V804L, V804M, G810S, and G810R. In someembodiments, the RET kinase protein point mutations/insertions/deletionsoccur in a RET fusion protein (e.g., any of the RET gene fusion proteinsdescribed in Table 1). In some embodiments, a compound of Formula I-IVis a polymorph form. In some embodiments, the compound is polymorph FormA of the compound of Formula I. In some embodiments, the compound of ispolymorph Form 1 of the compound of Formula II. In some embodiments, thecompound is polymorph Form 2 of the compound of Formula II. In someembodiments, the compound is polymorph Form 7 of the compound of FormulaII. In some embodiments, the compound is polymorph Form 8 of thecompound of Formula II. In some embodiments, the compound is polymorphForm A of the compound of Formula III. In some embodiments, the compoundis polymorph Form A of the compound of Formula IV. In some embodiments,the compound is polymorph Form B of the compound of Formula IV.

In some embodiments, the compound of Formula I-IV is a pharmaceuticallyacceptable salt. In some embodiments, the compound is a chloride salt ofthe compound of Formula I. In some embodiments, the compound is abromide salt of the compound of Formula I. In some embodiments, thecompound is an L-malate salt of the compound of Formula I. In someembodiments, the compound is a D-malate salt of the compound of FormulaI. In some embodiments, the compound is a phosphate salt of the compoundof Formula II. In some embodiments, the phosphate salt is asesqui-phosphate salt (e.g., 1.4:1, PO₄:free base).

In some embodiments of any of the methods or uses described herein, thecancer (e.g., RET-associated cancer) is a hematological cancer. In someembodiments of any of the methods or uses described herein, the cancer(e.g., RET-associated cancer) is a solid tumor (e.g., an advanced solidtumor and/or a RET-fusion positive solid tumor). In some embodiments ofany of the methods or uses described herein, the cancer (e.g.,RET-associated cancer) is a lung cancer (e.g., small cell lung carcinomaor non-small cell lung carcinoma), thyroid cancer (e.g., papillarythyroid cancer, medullary thyroid cancer (e.g., sporadic medullarythyroid cancer or hereditary medullary thyroid cancer), differentiatedthyroid cancer, recurrent thyroid cancer, or refractory differentiatedthyroid cancer), thyroid ademona, endocrine gland neoplasms, lungadenocarcinoma, bronchioles lung cell carcinoma, multiple endocrineneoplasia type 2A or 2B (MEN2A or MEN2B, respectively),pheochromocytoma, parathyroid hyperplasia, breast cancer, mammarycancer, mammary carcinoma, mammary neoplasm, colorectal cancer (e.g.,metastatic colorectal cancer), papillary renal cell carcinoma,ganglioneuromatosis of the gastroenteric mucosa, inflammatorymyofibroblastic tumor, or cervical cancer. In some embodiments of any ofthe methods or uses described herein, the cancer (e.g., RET-associatedcancer) is selected from the group of: acute lymphoblastic leukemia(ALL), acute myeloid leukemia (AML), cancer in adolescents,adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma,atypical teratoid/rhabdoid tumor, basal cell carcinoma, bile ductcancer, bladder cancer, bone cancer, brain stem glioma, brain tumor,breast cancer, bronchial tumor, Burkitt lymphoma, carcinoid tumor,unknown primary carcinoma, cardiac tumors, cervical cancer, childhoodcancers, chordoma, chronic lymphocytic leukemia (CLL), chronicmyelogenous leukemia (CML), chronic myeloproliferative neoplasms,neoplasms by site, neoplasms, colon cancer, colorectal cancer,craniopharyngioma, cutaneous T-cell lymphoma, cutaneous angiosarcoma,bile duct cancer, ductal carcinoma in situ, embryonal tumors,endometrial cancer, ependymoma, esophageal cancer,esthesioneuroblastoma, Ewing sarcoma, extracranial germ cell tumor,extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer,fallopian tube cancer, fibrous histiocytoma of bone, gallbladder cancer,gastric cancer, gastrointestinal carcinoid tumor, gastrointestinalstromal tumors (GIST), germ cell tumor, gestational trophoblasticdisease, glioma, hairy cell tumor, hairy cell leukemia, head and neckcancer, thoracic neoplasms, head and neck neoplasms, CNS tumor, primaryCNS tumor, heart cancer, hepatocellular cancer, histiocytosis, Hodgkin'slymphoma, hypopharyngeal cancer, intraocular melanoma, islet celltumors, pancreatic neuroendocrine tumors, Kaposi sarcoma, kidney cancer,Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oralcavity cancer, liver cancer, lung cancer, lymphoma, macroglobulinemia,malignant fibrous histiocytoma of bone, osteocarcinoma, melanoma, Merkelcell carcinoma, mesothelioma, metastatic squamous neck cancer, midlinetract carcinoma, mouth cancer, multiple endocrine neoplasia syndromes,multiple myeloma, mycosis fungoides, myelodysplastic syndromes,myelodysplastic/myeloproliferative neoplasms, neoplasms by site,neoplasms, myelogenous leukemia, myeloid leukemia, multiple myeloma,myeloproliferative neoplasms, nasal cavity and paranasal sinus cancer,nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-smallcell lung cancer, lung neoplasm, pulmonary cancer, pulmonary neoplasms,respiratory tract neoplasms, bronchogenic carcinoma, bronchialneoplasms, oral cancer, oral cavity cancer, lip cancer, oropharyngealcancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis,paraganglioma, paranasal sinus and nasal cavity cancer, parathyroidcancer, penile cancer, pharyngeal cancer, pheochromosytoma, pituitarycancer, plasma cell neoplasm, pleuropulmonary blastoma,pregnancy-associated breast cancer, primary central nervous systemlymphoma, primary peritoneal cancer, prostate cancer, rectal cancer,colon cancer, colonic neoplasms, renal cell cancer, retinoblastoma,rhabdomyosarcoma, salivary gland cancer, sarcoma, Sezary syndrome, skincancer, Spitz tumors, small cell lung cancer, small intestine cancer,soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer,stomach cancer, T-cell lymphoma, testicular cancer, throat cancer,thymoma and thymic carcinoma, thyroid cancer, transitional cell cancerof the renal pelvis and ureter, unknown primary carcinoma, urethralcancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer,and Wilms' tumor.

In some embodiments, a hematological cancer (e.g., hematological cancersthat are RET-associated cancers) is selected from the group consistingof leukemias, lymphomas (non-Hodgkin's lymphoma), Hodgkin's disease(also called Hodgkin's lymphoma), and myeloma, for instance, acutelymphocytic leukemia (ALL), acute myeloid leukemia (AML), acutepromyelocytic leukemia (APL), chronic lymphocytic leukemia (CLL),chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML),chronic neutrophilic leukemia (CNL), acute undifferentiated leukemia(AUL), anaplastic large-cell lymphoma (ALCL), prolymphocytic leukemia(PML), juvenile myelomonocyctic leukemia (JMML), adult T-cell ALL, AMLwith trilineage myelodysplasia (AML/TMDS), mixed lineage leukemia (MEL),myelodysplastic syndromes (MDSs), myeloproliferative disorders (MPD),and multiple myeloma (MM). Additional examples of hematological cancersinclude myeloproliferative disorders (MPD) such as polycythemia vera(PV), essential thrombocytopenia (ET) and idiopathic primarymyelofibrosis (IMF/IPF/PMF). In one embodiment, the hematological cancer(e.g., the hematological cancer that is a RET-associated cancer) is AMLor CMML.

In some embodiments, the cancer (e.g., the RET-associated cancer) is asolid tumor. Examples of solid tumors (e.g., solid tumors that areRET-associated cancers) include, for example, thyroid cancer (e.g.,papillary thyroid carcinoma, medullary thyroid carcinoma), lung cancer(e.g., lung adenocarcinoma, small-cell lung carcinoma), pancreaticcancer, pancreatic ductal carcinoma, breast cancer, colon cancer,colorectal cancer, prostate cancer, renal cell carcinoma, head and necktumors, neuroblastoma, and melanoma. See, for example, Nature ReviewsCancer, 2014, 14, 173-186.

In some embodiments, the cancer is selected from the group consisting oflung cancer, papillary thyroid cancer, medullary thyroid cancer,differentiated thyroid cancer, recurrent thyroid cancer, refractorydifferentiated thyroid cancer, multiple endocrine neoplasia type 2A or2B (MEN2A or MEN2B, respectively), pheochromocytoma, parathyroidhyperplasia, breast cancer, colorectal cancer, papillary renal cellcarcinoma, ganglioneuromatosis of the gastroenteric mucosa, and cervicalcancer.

In some embodiments, the patient is a human.

Compounds of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof are also useful for treating aRET-associated cancer.

Accordingly, also provided herein is a method for treating a patientdiagnosed with or identified as having a RET-associated cancer, e.g.,any of the exemplary RET-associated cancers disclosed herein, comprisingadministering to the patient a therapeutically effective amount of acompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, or a pharmaceutical compositionthereof as defined herein.

Dysregulation of a RET kinase, a RET gene, or the expression or activityor level of any (e.g., one or more) of the same can contribute totumorigenesis. For example, a dysregulation of a RET kinase, a RET gene,or expression or activity or level of any of the same can be atranslocation, overexpression, activation, amplification, or mutation ofa RET kinase, a RET gene, or a RET kinase domain. Translocation caninclude a gene translocation resulting in the expression of a fusionprotein that includes a RET kinase domain and a fusion partner. Forexample, a fusion protein can have increased kinase activity as comparedto a wildtype RET protein. In some embodiments, a mutation in a RET genecan involve mutations in the RET ligand-binding site, extracellulardomains, kinase domain, and in regions involved in protein:proteininteractions and downstream signaling. In some embodiments, a mutation(e.g., an activating mutation) in a RET gene can result in theexpression of a RET kinase having one or more (e.g., two, three, four,five, six, seven, eight, nine, or ten) amino acid substitutions (e.g.,one or more amino acid substitutions in the kinase domain (e.g., aminoacid positions 723 to 1012 in a wildtype RET protein), a gatekeeperamino acid (e.g., amino acid position 804 in a wildtype RET protein),the P-loop (e.g., amino acid positions 730-737 in a wildtype RETprotein), the DFG motif (e.g., amino acid positions 892-894 in awildtype RET protein), ATP cleft solvent front amino acids (e.g., aminoacid positions 758, 811, and 892 in a wildtype RET protein), theactivation loop (e.g., amino acid positions 891-916 in a wildtype RETprotein), the C-helix and loop preceeding the C-helix (e.g., amino acidpositions 768-788 in a wildtype RET protein), and/or the ATP bindingsite (e.g., amino acid positions 730-733, 738, 756, 758, 804, 805, 807,811, 881, and 892 in a wildtype RET protein). In some embodiments, amutation can be a gene amplification of a RET gene. In some embodiments,a mutation (e.g., an activating mutation) in a RET gene can result inthe expression of a RET kinase or RET receptor that lacks at least oneamino acid (e.g., at least 2, at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10, at least 12,at least 14, at least 16, at least 18, at least 20, at least 25, atleast 30, at least 35, at least 40, at least 45, or at least 50 aminoacids) as compared to a wildtype RET protein. In some embodiments,dyregulation of a RET kinase can be increased expression (e.g.,increased levels) of a wildtype RET kinase in a mammalian cell due toaberrant cell signaling and/or dysregulated autocrine/paracrinesignaling (e.g., as compared to a control non-cancerous cell). In someembodiments, a mutation (e.g., an activating mutation) in a RET gene canresult in the expression of a RET kinase or RET receptor that has atleast one amino acid (e.g., at least 2, at least 3, at least 4, at least5, at least 6, at least 7, at least 8, at least 9, at least 10, at least12, at least 14, at least 16, at least 18, at least 20, at least 25, atleast 30, at least 35, at least 40, at least 45, or at least 50 aminoacids) inserted as compared to a wildtype RET protein. In someembodiments, dyregulation of a RET kinase can be increased expression(e.g., increased levels) of a wildtype RET kinase in a mammalian cell(e.g., as compared to a control non-cancerous cell), e.g., due toaberrant cell signaling and/or dysregulated autocrine/paracrinesignaling. Other dysregulations can include RET mRNA splice variants. Insome embodiments, the wildtype RET protein is the exemplary wildtype RETprotein described herein.

In some embodiments, the dysregulation of a RET gene, a RET kinase, orexpression or activity or level of any of the same, includesoverexpression of wild-type RET kinase (e.g., leading to autocrineactivation). In some embodiments, the dysregulation of a RET gene, a RETkinase protein, or expression or activity or level of any of the same,includes overexpression, activation, amplification, or mutation in achromosomal segment comprising the RET gene or a portion thereof,including, for example, the kinase domain portion, or a portion capableof exhibiting kinase activity.

In some embodiments, the dysregulation of a RET gene, a RET kinaseprotein, or expression or activity or level of any of the same, includesone or more chromosome translocations or inversions resulting in a RETgene fusion. In some embodiments, the dysregulation of a RET gene, a RETkinase protein, or expression or activity or level of any of the same,is a result of genetic translocations in which the expressed protein isa fusion protein containing residues from a non-RET partner protein, andincludes a minimum of a functional RET kinase domain.

Non-limiting examples of RET fusion proteins are shown in Table 1.

TABLE 1 Exemplary RET Fusion Partners and Cancers Non-limiting ExemplaryFusion Partner RET-Associated Cancer(s) BCR Chronic MyelomonocyticLeukemia (CMML) CLIP1 Adenocarcinoma KIF5B NSCLC, Ovarian Cancer,Spitzoid Neoplasms; Lung Adenocarcinoma^(3,4,14,28); AdenosquamousCarcinomas¹⁵ CCDC6 (also NSCLC, Colon Cancer, called PTC1, PapillaryThyroid Cancer; D10S170, or H4) Adenocarcinomas; Lung Adenocarcinoma;Metastatic Colorectal Cancer⁵; Adenosquamous Carcinomas¹⁵, BreastCancer³⁰ PTC1ex9 (a novel Metastatic papillary thyroid CCDC6 cancer²rearrangement) NCOA4 (also Papillary Thyroid Cancer²¹, called PTC3,NSCLC, Colon Cancer, ELE1, and RFG) Salivary Gland Cancer, MetastaticColorectal Cancer⁵; Lung Adenocarcinoma¹⁵; Adenosquamous Carcinomas¹⁵Diffuse Sclerosing Variant of Papillary Thyroid Cancer¹⁶, BreastCancer³⁰, Acinic Cell Carcinoma³², Mammary Analog Secretory Carcinoma³³TRIM33 (also NSCLC, Papillary Thyroid called PTC7, Cancer, Lung RFG7,and TIF1G) Adenocarcinoma⁴⁶, Various²² ERC1 (also called PapillaryThyroid Cancer, ELKS and Breast Cancer RAB61P2) FGFR1OP CMML, PrimaryMyelofibrosis with secondary Acute Myeloid Leukemia MBD1(also knownPapillary Thyroid Cancer as PCM1) PRKAR1A (also Papillary Thyroid Cancercalled PTC2) TRIM24 (also Papillary Thyroid Cancer called PTC6) KTN1(also called Papillary Thyroid Cancer PTC8) GOLGA5 (also PapillaryThyroid Cancer, called PTC5) Spitzoid Neoplasms HOOK3 Papillary ThyroidCancer KIAA1468 (also Papillary Thyroid Cancer, called PTC9 and LungAdenocarcinoma^(8,12) RFG9) TRIM27 (also Papillary Thyroid Cancer calledRFP) AKAP13 Papillary Thyroid Cancer FKBP15 Papillary Thyroid Cancer,Acute Myeloid Leukemia⁴⁶ SPECC1L Papillary Thyroid Cancer; Thyroid GlandCarcinoma TBL1XR1 Papillary Thyroid Cancer; Thyroid Gland CarcinomaCEP55 Diffuse Gastric Cancer⁷ CUX1 Lung Adenocarcinoma ACBD5 PapillaryThyroid Carcinoma MYH13 Medullary Thyroid Carcinoma¹ UncharacterizedInflammatory Myofibroblastic Tumor⁶ PIBF1 Bronchiolus Lung CellCarcinoma⁹ KIAA1217 (also Papillary Thyroid Cancer^(10,13) called SKT)Lung Adenocarcinoma¹⁴ NSCLC¹⁴ MPRIP NSCLC¹¹ HRH4-RET Thyroid Cancerand/or Paillary Thyroid Carcinoma¹⁷ Ria-RET Thyroid Cancer and/orPapillary Thyroid Carcinoma¹⁷ RFG8 Papillary Thyroid Carcinoma¹⁸ FOXP4Lung Adenocarcinoma¹⁹ MYH10 Infantile Myofibromatosis²⁰ HTIF1 Various²²H4L Various²² PTC4 (a novel Papillary Thyroid Cancer²³ NCO4/ELE1rearrangement) FRMD4A NSCLC²⁴ SQSTM1 Papillary Thyroid Carcinoma²⁵AFAP1L2 Papillary Thyroid Carcinoma²⁵ AFAP1 NSCLC³¹ PPFIBP2 PapillaryThyroid Carcinoma²⁵ EML4 NSCLC PARD3 NSCLC²⁷ RASGEF1A Breast Cancer³⁰TEL (also called In vitro³⁴, secretory ETV6) carcinoma⁵¹ RUFY1Colorectal Cancer³⁵ OLFM4 Small-Bowel Cancer³⁶ UEVLD Papillary ThyroidCarcinoma²⁹ DLG5 Non-Anaplastic Thyroid (NAT) Cancer³⁷ RRBP1 ColonCancer³⁸ ANK3 Papillary Thyroid Carcinoma³⁹ PICALM NSCLC⁴⁰ MYO5C NSCLC⁴¹EPHA5 NSCLC⁴⁰ RUFY2 Lung Cancer⁴² KIF13A Lung Adenocarcinoma⁴³, NSCLC⁴⁵TNIP1 Colorectal Cancer⁴⁴ SNRNP70 Colorectal Cancer⁴⁴ MRLN ThyroidCarcinoma⁴⁶ LMNA Spitzoid Melanoma⁴⁷ RUFY3 Papillary Thyroid CarcinomaTFG MYO5A Pigmented spindle cell nevus (PSCN) of Reed⁴⁸ ADD3 Lungadenocarcinoma⁴⁹ JMJD1C NSCLC⁵⁰ RBPMS DOCK1 TAF3 NCOA1 NSCLC⁵² ¹Grubbset al., J. Clin. Endocrinol. Metab. 100: 788-793, 2015. ²Halkova et al.,Human Pathology 46: 1962-1969, 2015. ³U.S. Pat. No. 9,297,011 ⁴U.S. Pat.No. 9,216,172 ⁵Le Rolle et al., Oncotarget. 6(30): 28929-37, 2015.⁶Antonescu et al., Am J Surg Pathol. 39(7): 957-67, 2015. ⁷U.S. patentapplication Pub. No. 2015/0177246. ⁸U.S. patent application Pub. No.2015/0057335. ⁹Japanese Patent Application Publication No. 2015/109806A.¹⁰Chinese Patent Application Publication No. 105255927A. ¹¹Fang, et al.Journal of Thoracic Oncology 11.2 (2016): S21-S22. ¹²European PatentApplication Publication No. EP3037547A1. ¹³Lee et al., Oncotarget. DOI:10.18632/oncotarget.9137, e-published ahead of printing, 2016. ¹⁴Saitoet al., Cancer Science 107: 713-720, 2016. ¹⁵Pirker et al., Transl. LungCancer Res. 4(6): 797-800, 2015. ¹⁶Joung et al., Histopathology 69(1):45-53, 2016. ¹⁷PCT patent application Pub. No. WO 2016/141169.¹⁸Klugbauer et al., Cancer Res., 60(24): 7028-32, 2000. ¹⁹Bastien etal., Journal of Molecular Diagnostics, 18(6): 1027, Abstract Number:S120, 2016 Annual Meeting of the Association for Molecular Pathology,Charlotte, NC, 2016. ²⁰Rosenzweig et al., Pediatr Blood Cancer, doi:10.1002/pbc.26377, 2016. ²¹Su et al., PLoS One, 11(111): e0165596, 2016.²²U.S. Pat. No. 9,487,491. ²³Fugazzola et al., Oncogene, 13(5): 1093-7,1996. ²⁴Velcheti et al., J Thorac Oncol., 12(2): e15-e16. doi:10.1016/j.jtho.2016.11.274, 2017. ²⁵Kato et al, Clin Cancer Res. 2017Apr. 15; 23(8): 1988-1997. doi: 10.1158/1078-0432.CCR-16-1679. Epub 2016Sep. 28. ²⁶ Drilon, Alexander, et al. “A phase 1/1b study of RXDX-105,an oral RET and BRAF inhibitor, in patients with advanced solid tumors.”Aug. 8, (2016): 7. ²⁷Sabari et al., Oncoscience, Advance Publications,www.impactjournals.com/oncoscience/files/papers/1/345/345.pdf, 2017.²⁸U.S. patent application Pub. No. 2017/0014413. ²⁹Lu et al.,Oncotarget, 8(28): 45784-45792, doi: 10.18632/oncotarget.17412, 2017.³⁰Hirshfield et al., Cancer Research, (February 2017) Vol. 77, No. 4,Supp. 1. Abstract Number: P3-07-02. Meeting Info: 39th Annual CTRC-AACRSan Antonio Breast Cancer Symposium. San Antonio, TX, United States. 6Dec. 2016-10 Dec. 2016. ³¹Morgensztern et al., Journal of ThoracicOncology, (January 2017) Vol. 12, No. 1, Supp. 1, pp. S717-S718,Abstract Number: P1.07-035, Meeting Info: 17th World Conference of theInternational Association for the Study of Lung Cancer, IASLC 2016.Vienna, Austria. 4 Dec. 2016. ³²Dogan et al., Laboratory Investigation,(February 2017) Vol. 97, Supp. 1, pp. 323A. Abstract Number: 1298,Meeting Info: 106th Annual Meeting of the United States and CanadianAcademy of Pathology, USCAP 2017. San Antonio, TX, United States.³³Dogan et al., MODERN PATHOLOGY, Vol. 30, Supp. [2], pp. 323A-323A. MA1298, 2017. ³⁴PCT patent application Pub. No. WO 2017/146116. ³⁵PCTpatent application Pub. No. WO 2017/122815. ³⁶Reeser et al., J. Mol.Diagn., 19(5): 682-696, doi: 10.1016/j.jmoldx.2017.05.006, 2017.³⁷Ibrahimpasic et al., Clin. Cancer Res., doi:10.1158/1078-0432.CCR-17-1183, 2017. ³⁸Kloosterman et al., Cancer Res.,77(14): 3814-3822. doi: 10.1158/0008-5472.CAN-16-3563, 2017. ³⁹Chai etal., Oncology Reports, 35(2): 962-970. doi: 10.3892/or.2015.4466, 2015.⁴⁰Gautschi et al. Journal of Clinical Oncology, 35(13) 1403-1410. doi:10.1200/JCO.2016.70.9352, 2017. ⁴¹Lee et al. Annals of Oncology, 28(2),292-297. doi: 10.1093/annonc/mdw559, 2016. ⁴²Zheng et al. NatureMedicine, 20(12), 1479-1484. doi: 10.1038/nm.3729, 2014. ⁴³Zhang et al.Lung Cancer, 118, 27-29. doi: 10.1016/j.lungcan.2017.08.019, 2018.⁴⁴Morano et al. Molecular Cancer Therapeutics, (January 2018) Vol. 17,No. 1, Supp. Supplement 1. Abstract Number: B049. Meeting Info:AACR-NCI-EORTC International Conference: Molecular Targets and CancerTherapeutics 2017. ⁴⁵Wang et al. Journal of Thoracic Oncology, (November2017) Vol. 12, No. 11, Supp. Supplement 2, pp. S2105. Abstract Number:P2.02-018. Meeting Info: 18th World Conference on Lung Cancer of theInternational Association for the Study of Lung Cancer, IASLC 2017.Yokohama, Japan. 15 Oct. 2017-18 Oct. 2017. ⁴⁶Gao et al. Cell Reports,23(1), 227-238. doi: 10.1016/j.celrep.2018.03.050, 2018. ⁴⁷U.S. patentapplication Pub. No. 2016/0010068. ⁴⁸VandenBoom, et al. Am. J. Surg.Pathol. 42(8): 1042-1051, 2018. doi: 10.1097/PAS.0000000000001074 ⁴⁹Cao,et al. Onco. Targets. Ther. 2018(11): 2637-2646, 2018. doi:10.2147/OTT.S155995 ⁵⁰Luo, et al. Int. J. Cancer, 2018. epub ahead ofprint, doi: 10.1002/ijc.31542 ⁵¹Guilmette, et al. Hum Pathol. pii:S0046-8177(18)30316-2, 2018. doi: 10.1016/j.humpath.2018.08.011 ⁵²Zhao,et al. Journal of Clinical Oncology Vol 36, No. 15, Supp. [S], MAe21139.

In some embodiments, the dysregulation of a RET gene, a RET kinase, orexpression or activity or level of any of the same, includes one or moredeletions (e.g., deletion of an amino acid at position 4), insertions,or point mutation(s) in a RET kinase. In some embodiments, thedysregulation of a RET gene, a RET kinase, or expression or activity orlevel of any of the same, includes a deletion of one or more residuesfrom the RET kinase, resulting in constitutive activity of the RETkinase domain.

In some embodiments, the dysregulation of a RET gene, a RET kinase, orexpression or activity or level of any of the same, includes at leastone point mutation in a RET gene that results in the production of a RETkinase that has one or more amino acid substitutions, insertions, ordeletions as compared to the wild-type RET kinase (see, for example, thepoint mutations listed in Table 2).

TABLE 2 RET Kinase Protein Amino AcidSubstitutions/Insertions/Deletions^(A) Amino acid position 2 Amino acidposition 3 Amino acid position 4 Amino acid position 5 Amino acidposition 6 Amino acid position 7 Amino acid position 8 Amino acidposition 11 Amino acid position 12 Amino acid position 13 Amino acidposition 20 Amino acid position 32 (e.g., S32L) Amino acid position 34(e.g., D34S) Amino acid position 40 (e.g., L40P) Amino acid position 45(e.g., A45A)³⁹ Amino acid position 56 (e.g., L56M)³⁰ Amino acid position64 (e.g., P64L) Amino acid position 67 (e.g., R67H) Amino acid position77 (e.g., R77C)⁶⁵ Amino acid position 114 (e.g., R114H) Amino acidposition 136 (e.g., glutamic acid to stop codon) Amino acid position 145(e.g., V145G) Amino acid position 177 (e.g., R177L)⁶⁷ Amino acidposition 180 (e.g., arginine to stop codon) Amino acid position 200Amino acid position 270 (e.g., P270L)⁶⁵ Amino acid position 278 (e.g.,T278N)⁵⁷ Amino acid position 292 (e.g., V292M) Amino acid position 294Amino acid position 321 (e.g., G321R) Amino acid position 330 (e.g.,R330Q) Amino acid position 338 (e.g., T338I) Amino acid position 360(e.g., R360W) Amino acid position 373 (e.g., alanine to frameshift) ΔAmino acid positions 378-385 with insertion of one amino acid (e.g.,D378-G385 > E) Amino acid position 393 (e.g., F393L) Amino acid position423 (e.g., G423R)²⁷ Amino acid position 428 (e.g., E428K)⁵⁷ Amino acidposition 432 (e.g., A432A³⁹) Amino acid position 446 (e.g., G446R)²⁸ ΔAmino acid positions 505-506 (6-Base Pair In-Frame Germline Deletion inExon 7)³ Amino acid position 510 (e.g., A510V) Amino acid position 511(e.g., E511K) Amino acid position 513 (e.g., G513D)⁷* Amino acidposition 515 (e.g., C515S, C515W⁴) Amino acid position 525 (e.g.,R525W)⁷* Amino acid position 531 (e.g., C531R, or 9 base pairduplication²) Amino acid position 532 (e.g., duplication)² Amino acidposition 533 (e.g., G533C, G533S) Amino acid position 534 (e.g., L534L)⁶Amino acid position 550 (e.g., G550E) Amino acid position 591 (e.g.,V591I) Amino acid position 593 (e.g., G593E) Amino acid position 595(e.g., E595D and E595A)¹⁸ Amino acid position 600 (e.g., R600Q) Aminoacid position 602 (e.g., I602V)⁶ Amino acid position 603 (e.g., K603Q,K603E²) Amino acid position 606 (e.g., Y606C) Amino acid position 609(e.g., C609Y, C609S, C609G, C609R, C609F, C609W, C609C³²) Amino acidposition 611 (e.g., C611R, C611S, C611G, C611Y, C611F, C611W) Amino acidposition 616 (e.g., E616Q)²³ Δ Amino acid position 616⁶⁴ Amino acidposition 618 (e.g., C618S, C618Y, C618R, C618G, C618F, C618W, stop⁵⁶)Amino acid position 619 (e.g., F619F) Amino acid position 620 (e.g.,C620S, C620W, C620R, C620G, C620L, C620Y, C620F, C620A⁴⁷) Δ Amino acidpositions 612-620⁷⁴ Amino acid position 622 (e.g., P622L)⁶⁸ Amino acidposition 623 (e.g., E623K) Amino acid position 624 (e.g., D624N) Aminoacid position 628 (e.g., P628N)⁷³ Amino acid positions 629-631 (e.g.,L629- D631delinsH)⁸⁰ Amino acid position 630 (e.g., C630A, C630R, C630S,C630Y, C630F, C630W) Δ Amino acid position 630⁵⁶ Amino acid position 631(e.g., D631N, D631Y, D631A, D631G, D631V, D631E,) Δ Amino acid position631⁶⁹ Amino acid positions 631-633 > V (i.e., residues 631- 633 arereplaced with a single valine residue) Amino acid positions 631-633 > A(i.e., residues 631- 633 are replaced with a single alanine residue)Amino acid positions 631-633 > E (i.e., residues 631- 633 are replacedwith a single glutamic acid residue) Δ Amino acid positions 631-633(e.g., D631-L633) Δ Amino acid positions 631-634 (e.g., D631-C634) Aminoacid position 632 (e.g., E632K, E632G^(5,11), E632V⁶², 632 toframeshift⁴⁷) Amino acid positions 632-633 > V (i.e., residues 632 and633 are replaced with a single valine residue)⁷⁴ Δ Amino acid positions632-633 (e.g., E632-L633 in either the somatic cells, or a 6-Base PairIn-Frame Germline Deletion in Exon 11⁹) Amino acid positions 632-639 >HR (i.e., residues 632- 639 are replaced with two residues, histidineand arginine) Amino acid position 633 (e.g., L633R⁶², 9 base pairduplication², L633delinsLCR⁷¹) Amino acid position 634 (e.g., C634W,C634Y, C634S, C634R, C634F, C634G, C634L, C634A, or C634T, a 9 base pairdeletion⁶², a 9 base pair duplication⁵⁶, or a 12 base pair duplication²)(e.g., causing MTC) Δ Amino acid position 634⁵⁶ Amino acid position632/633/634 (E632V/L633R/634 9 base pair deletion)⁶² Amino acid position635 (e.g., R635G or an insertion ELCR²) Amino acid position 636 (e.g.,T636P², T636M⁴) Amino acid positions 636-637 (e.g., T636- V637insCRT)⁸⁰Amino acid position 638 (e.g., isoleucine to frameshift⁴⁷) Amino acidposition 640 (e.g., A640G) Amino acid position 634/640 (e.g.,C634R/A640G)⁵⁶ Amino acid position 641 (e.g., A641S, A641T⁸) Amino acidposition 634/641 (e.g., C634S/A641S)⁵⁶ Amino acid position 639/641(e.g., A639G/A641R)⁵⁶ Amino acid position 644 (e.g., T644M)⁵⁹ Amino acidposition 648 (e.g., V648I) Amino acid positions 634/648 (e.g.,C634R/V648I)⁷⁷ Amino acid position 649 (e.g., S649L)²⁸ Amino acidposition 661 (e.g., H661H)⁶ Amino acid position 664 (e.g., A664D) Aminoacid position 665 (e.g., H665Q) Amino acid position 666 (e.g., K666E,K666M, K666N, K666R) Amino acid position 675 (T675T, silent nucleotidechange)¹⁸ Amino acid position 679 (e.g., P679P)⁶ Amino acid position 680(e.g., A680T, alanine to frameshift)⁶ Amino acid position 686 (e.g.,S686N) Amino acid position 689 (e.g., S689T)¹⁸ Amino acid position 691(e.g., G691S) Amino acid position 694 (e.g., R694Q) Amino acid position700 (e.g., M700L) Amino acid position 706 (e.g., V706M, V706A) Aminoacid position 713 splice variant (e.g., E713K (e.g., a splice variant))⁶Amino acid position 714 (e.g., D714Y)⁵⁷ Amino acid position 727 (e.g.,G727E)⁶ Amino acid position 732 (e.g., E732K)²⁰ Amino acid position 734(e.g., E734K)⁴⁸ Amino acid position 736 (e.g., G736R)⁶ Amino acidposition 738 (e.g., V738V)⁶ Amino acid position 742 (e.g., T742M)⁵¹Amino acid position 748 (e.g., G748C) Amino acid position 749 (e.g.,R749T³⁶) Amino acid position 750 (e.g., A750P, A750G⁶) Amino acidposition 752 (e.g., Y752Y)⁶ Amino acid position 751 (e.g., G751G)⁶ Aminoacid position 762 (e.g., E762Q³⁶) Amino acid position 765 (e.g., S765P,S765F) Amino acid position 766 (e.g., P766S, P766M⁶) Amino acid position768 (e.g., E768Q, E768D, E768N⁴⁶, E768G⁷²) Amino acid position 769(e.g., L769L⁶) Amino acid position 770 (e.g., R770Q) Amino acid position771 (e.g., D771N) Amino acid position 777 (e.g., N777S) Amino acidposition 778 (e.g., V778I) Amino acid position 781 (e.g., Q781R) Aminoacid position 788 (e.g., I788I³², I788N⁷⁸) Amino acid position 790(e.g., L790F) Amino acid position 768/790 (e.g., E768D/L790T)⁴⁰ Aminoacid position 791 (e.g., Y791F, Y791N²⁴) Amino acid position 634/791(e.g., C634Y/Y791F)⁵⁵ Amino acid position 790/791 (e.g., L790F/Y791F)⁵⁵Amino acid position 802 Amino acid position 804 (e.g., V804L^(15,16),V804M^(15,16), V804E¹²) (e.g., causing MTC) Amino acid position778/804⁵⁰ (e.g., V778I/V804M⁵⁴) Amino acid position 781/804 (e.g.,Q781R/V804M)⁴¹ Amino acid position 805 (e.g., E805K) Amino acid position804/805 (e.g., V804M/E805K)¹⁷ Amino acid position 806 (e.g., Y806F,Y806S¹², Y806G, Y806C^(2,12,14), Y806E¹⁴, Y806H¹², Y806N^(12,) Y806Y³²)Amino acid position 804/806 (e.g., V804M/Y806C)³⁸ Amino acid position810 (e.g., G810R¹², G810S¹², G810A¹³, G810C, G810V, and G810D) Aminoacid position 818 (e.g., E818K) Amino acid position 819 (e.g., S819I)Amino acid position 820 (e.g., R820L)⁵⁷ Amino acid position 823 (e.g.,G823E) Amino acid position 826 (e.g., Y826M, Y826S)¹⁰ Amino acidposition 828 (e.g., G828R)⁵⁷ Amino acid position 833 (e.g., R833C) Aminoacid position 836 (e.g., S836S)¹⁹ Amino acid position 841 (e.g., P841L,P841P) Amino acid position 843 (e.g., E843D) Amino acid position 844(e.g., R844W, R844Q, R844L) Amino acid position 804/844 (e.g.,V804M/R844L)⁷⁶ Amino acid position 845 (e.g., A845A)⁶³ Amino acidposition 848 (e.g., M848T) Amino acid position 852 (e.g., I852M) Aminoacid position 853 (e.g., S853T)⁵⁷ Amino acid position 865 (e.g.,L865V)¹² Amino acid position 866 (e.g., A866W)³³ Amino acid position 867(e.g., E867K)³⁷ Amino acid position 870 (e.g., L870F)¹² Amino acidposition 873 (e.g., R873W, R873Q⁴²) Amino acid position 876 (e.g.,A876V) Amino acid position 881 (e.g., L881V) Amino acid position 882Amino acid position 883 (e.g., A883F, A883S, A883T, A883Y⁵³, A883V)Amino acid position 884 (e.g., E884K, E884V³⁵) Amino acid position 886(e.g., R886W) Amino acid position 891 (e.g., S891A, S891S³², S891L³⁵)Amino acid position 893 (e.g., F893L)⁴² Amino acid position 894 (e.g.,G894S)⁴³ Amino acid position 897 (e.g., R897Q, R897P) Amino acidposition 898 (e.g., D898V, D898Y⁶⁶) Δ Amino acid position 898 Δ Aminoacid positions 898-902⁵⁸ Δ Amino acid positions 899-902⁴⁷ Δ Amino acidpositions 898-901⁴⁷ Δ Amino acid positions 632-633/Δ Amino acidpositions 898-901⁴⁷ Amino acid position 900 (e.g., Y900F)²² Amino acidposition 901 (e.g., E901K) Amino acid position 904 (e.g., S904F, S904S,S904C², S904T⁵⁷) Amino acid position 691/904 (e.g., G691S/S904S)⁴⁹ Aminoacid position 804/904 (e.g., V804M/S904C)³⁸ Amino acid position 905(e.g., Y905F)²² Amino acid position 907 (e.g., K907E, K907M) Amino acidposition 908 (e.g., R908K) Amino acid position 911 (e.g., G911D, G911G(e.g., a splice variant)⁶) Amino acid position 912 (e.g., R912P, R912Q)Amino acid position 918 (e.g., M918T², M918V, M918L⁶) (e.g., causingMTC) Amino acid position 591/918 (e.g., V591I/M918T)⁶¹ Amino acidposition 620/918 (e.g., C620F/M918T)⁴⁷ Amino acid position 891/918(e.g., S891A/M918T)⁴⁷ Δ Amino acid position 898-901/M918T⁴⁷ Amino acidposition 919 (e.g., A919V, A919P⁵²) Amino acid position 768/919⁵⁴ Aminoacid position 921 (e.g., E921K, E921D) Amino acid position 911/918/921(e.g., G911E/M918T/E921K)⁶¹ Amino acid position 922 (e.g., S922P, S922Y)Amino acid position 924 (e.g., F924S)⁶ Amino acid position 930 (e.g.,T930M) Amino acid position 961 (e.g., F961L) Amino acid position 972(e.g., R972G) Amino acid position 973 (e.g., P973T)⁵⁷ Amino acidposition 977 (e.g., S977R)³⁷ Amino acid position 981 (e.g., Y981F)²²Amino acid position 982 (e.g., R982C)⁷⁰ Amino acid position 634/691/982(e.g., C634R/G691S/R982C)⁴⁵ Amino acid position 292/67/982 (e.g., V292M/R67H/R982C)⁷⁵ Amino acid position 634/292/67/982 (e.g., C634R/V292M/R67H/R982C)⁷⁵ Amino acid position 1009 (e.g., M1009V) Amino acidposition 1015 (e.g., Y1015F)²² Amino acid position 1017 (e.g., D1017N)Amino acid position 1024 (e.g., S1024F)⁷⁹ Amino acid position 1041(e.g., V1041G) Amino acid position 1047 (e.g., P1047S)⁶⁵ Amino acidposition 1051 (e.g., A1051T)⁵⁷ Δ Amino acid position 1059⁵⁷ Amino acidposition 1064 (e.g., M1064T) Amino acid position 1096 (e.g., Y1096F)²¹Amino acid position 1105 (e.g., A1105V)⁵⁷ Amino acid position 1109(e.g., M1109T)³⁴ RET + 3¹ (In-Frame Deletion in Exons 6 and 11)²⁵ (3bpIn-Frame Deletion in Exon 15)²⁶ Nucleotide position 2136+2 (e.g.,2136+2T > G)²⁹ (del632-636 ins6)³¹ Amino acid positions 791 and 852(e.g., Y791F + I852M)³¹ Amino acid positions 634 and 852 (e.g., C634R +I852M)³¹ c.1893_1895del⁴⁴ ^(A)The RET kinase mutations shown may beactivating mutations and/or confer increased resistance of the RETkinase to a RET kinase inhibitor and/or a multi-kinase inhibitor (MKI),e.g., as compared to a wildtype RET kinase. ¹U.S. patent applicationPub. No. 2014/0272951. ²Krampitz et al., Cancer 120: 1920-1931, 2014.³Latteyer, et al., J. Clin. Endocrinol. Metab. 101(3): 1016-22, 2016.⁴Silva, et al. Endocrine 49.2: 366-372, 2015. ⁵Scollo, et al., Endocr.J. 63(1): 87-91, 2016. ⁶Jovanovic, et al., Prilozi 36(1): 93-107, 2015.⁷Qi, et al., Oncotarget. 6(32): 33993-4003, 2015. *R525W and G513Dappear to act in combination with S891A to enchance oncogenic activity.⁸Kim, et al. ACTA ENDOCRINOLOGICA-BUCHAREST 11.2, 189-194, 2015.⁹Cecchirini, et al. Oncogene, 14, 2609-2612, 1997. ¹⁰Karrasch, et al.Eur. Thyroid J., 5(1): 73-7, 2016. ¹¹Scollo et al., Endocr. J. 63:87-91, 2016. ¹²PCT patent application Pub. No. WO 2016/127074. ¹³Huanget al., Mol. Cancer Ther., 2016 Aug. 5. pii: molcanther.0258.2016. [Epubahead of print]. ¹⁴Carlomagno, et al., Endocr. Rel. Cancer 16(1):233-41, 2009. ¹⁵Yoon et al., J. Med. Chem. 59(1): 358-73, 2016. ¹⁶U.S.Pat. No. 8,629,135. ¹⁷Cranston, et al., Cancer Res. 66(20): 10179-87,2006. ¹⁸Kheiroddin et al., Clin. 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S, pp. SUN-0322, Meeting Info.: 96thAnnual Meeting and Expo of the Endocrine-Society, Chicago, IL, USA, Jun.21-24, 2014. ³¹Gazizova et al., Endocrine Reviews, (JUNE 2014) Vol. 35,No. 3, Suppl. S, pp. SAT-0304, Meeting Info.: 96th Annual Meeting andExpo of the Endocrine-Society, Chicago, IL, USA, Jun. 21-24, 2014.³²Sromek et al., Endocr Pathol., doi: 10.1007/s12022-017-9487-2, 2017.³³U.S. patent application Pub. No. 2017/0267661. ³⁴Davila et. al., RareTumors, 2017; 9(2): 6834. doi: 10.4081/rt.2017.6834. ³⁵U.S. patentapplication Pub. No. 2018/0009818. ³⁶PCT patent application Pub. No. WO2017/197051 ³⁷European Patent Application Publication No. 3271848³⁸Roskoski and Sadeghi-Nejad, Pharmacol. Res., 128, 1-17. doi:10.1016/j.phrs.2017.12.021, 2018. ³⁹Kaczmarek-Ryś, et al.Endocrine-related cancer 25(4): 421-436. doi: 10.1530/ERC-17-0452, 2018.⁴⁰Raue, et al. J. Clin Endocrinol Metab, 103(1): 235-243. doi:10.1210/jc.2017-01884, 2018. ⁴¹Nakao, et al. Head and Neck, 35:E363-E368. doi: 10.1002/hed.23241, 2013. ⁴²Attié, et al. Human MolecularGenetics 4(8): 1381-1386. doi: 10.1093/hmg/4.8.1381, 1995. ⁴³Fitze, etal. Lancet, 393(9313): 1200-1205. doi: 10.1016/S0140-6736(02)08218-1,2002. ⁴⁴Weng, et al. Zhonghua Nei Ke Za Zhi, 57(2): 134-137. doi:10.3760/cma.j.issn.0578-1426.2018.02.010, 2018. ⁴⁵Chen, et al. MedicalJournal of Chinese People's Liberation Army 38.4 (2013): 308-312.⁴⁶Gudernova, et al. eLife, 6: e21536. doi: 10.7554/eLife.21536, 2017.⁴⁷Romei, et al. Oncotarget, 9(11): 9875-9884. doi:10.18632/oncotarget.23986, 2018. ⁴⁸Plaza-Menacho. Endocr Relat Cancer,25(2): T79-T90. doi: 10.1530/ERC-17-0354, 2017. ⁴⁹Guerin, et al. EndocrRelat Cancer, 25(2): T15-T28. doi: 10.1530/ERC-17-0266, 2017. ⁵⁰Roy etal. Oncologist, 18(10): 1093-1100. doi: 10.1634/theoncologist.2013-0053,2013 ⁵¹U.S. patent application Pub. No. 2017/0349953 ⁵²Santoro, et al.Endocrinology, 145(12), 5448-5451, 2004. doi: 10.1210/en.2004-0922⁵³U.S. Pat. No. 9,006,256 ⁵⁴Yeganeh, et al. Asian Pac J Cancer Prev,16(6), 2107-17. doi: 10.7314/APJCP.2015.16.6.2107 ⁵⁵Mulligan, L. M,Nature Reviews Cancer, 14(3), 173, 2014, doi: 10.1038/nrc3680 ⁵⁶Arighi,et al, Cytokine & Growth Factor Reviews, 16(4-5), 441-467, 2005. doi:10.1016/j.cytogfr.2005.05.010 ⁵⁷Dabir, et al, Journal of ThoracicOncology, 9(9), 1316-1323, 2014. doi: 10.1097/JTO.0000000000000234⁵⁸Uchino, et al, Cancer Science, 90(11), 1231-1237, 1999. doi:10.1111/j.1349-7006.1999.tb00701.x ⁵⁹Krampitz. Cancer, 120(13),1920-1931, 2014: 10.1002/cncr.28661 ⁶⁰ Jhiang et al, Thyroid 6(2), 1996.doi: 10.1089/thy.1996.6.115 ⁶¹Dvo{hacek over (r)}áková, et al, Thyroid,16(3), 311-316, 2006. doi: 10.1089/thy.2006.16.311 ⁶²Severskaya et al,Genomics Transcriptomics Proteomics, 40(3) 425-435. ⁶³Elisei, et al,Journal of Genetic Syndromes & Gene Therapy, 5(1), 1, 2014. doi:10.4172/2157-7412.1000214 ⁶⁴Ahmed et al, The Journal of MolecularDiagnostics, 7(2), 283-288, 2005. doi: 10.1016/S1525-1578(10)60556-9⁶⁵Oliveira, et al. J. Exp. Clin. Cancer Res. 37(84), 2018. doi:10.1186/s13046-018-0746-y ⁶⁶Yi, et al. Case Rep. Endocrinol. 2018:8657314, 2018. doi: 10.1155/2018/8657914 ⁶⁷Huang, et al. Cell. 173(2):355-370, 2018. doi: 10.1016/j.cell.2018.03.039 ⁶⁸Bosic, et al.Pathology. 50(3): 327-332, 2018. doi: 10.1016/j.pathol.2017.10.011⁶⁹Yao, et al. Zhonghua Yi Xue Za Zhi. 87(28): 1962-1965, 2007. PMID:17923033 ⁷⁰Quintela-Fandino, et al. Mol. Oncol. 8(8): 1719-1728, 2014.doi: 10.1016/j.molonc.2014.07.005 ⁷¹Urbini, et al. Int J Genomics 2018:6582014. doi: 10.1155/2018/6582014 ⁷²Yu, et al. Clin Lung Cancer, pii:S1525-7304(18)30204-3, 2018. doi: 10.1016/j.cllc.2018.08.010⁷³Soca-Chafre, et al. Oncotarget 9(55): 30499-30512, 2018. doi:10.18632/oncotarget.25369 ⁷⁴Kim, et al. BMC Urol 18(1): 68, 2018. doi:10.1186/s12894-018-0380-1 ⁷⁵Qi, et al. PLoS One 6(5): e20353, 2011. doi:10.1371/journal.pone.0020353 ⁷⁶Bartsch, et al Exp Clin EndocrinolDiabetes 108(2): 128-132, 2000. doi: 10.1055/s-2000-5806 ⁷⁷Nunes, et al.J Clin Endocrinol Metab. 87(12): 5658-5661, 2002. doi:10.1210/jc.2002-020345 ⁷⁸Plenker et al., Sci. Transl. Med., 9(394), doi:10.1126/scitranslmed.aah6144, 2017 ⁷⁹Romei, et al., European ThyroidJournal, Vol. 7, Supp. 1, pp 63. Abstract No: P1-07-69. Meeting Info:41st Annual Meeting of the European Thyroid Association, ETA 2018. 15Sep. 2018-18 Sep. 2018. doi: 10.1159/000491542 ⁸⁰Ciampi, et al.,European Thyroid Journal, Vol. 7, Supp. 1, pp 63. Abstract No: OP-09-66.Meeting Info: 41st Annual Meeting of the European Thyroid Association,ETA 2018. 15 Sep. 2018-18 Sep. 2018. doi: 10.1159/000491542

In some embodiments, the dysregulation of a RET gene, a RET kinase, orexpression or activity or level of any of the same, includes at leastone point mutation in a RET gene that results in the production of a RETkinase that has one or more amino acid substitutions, insertions, ordeletions as compared to the wild-type RET kinase (see, for example, thepoint mutations listed in Table 2a).

TABLE 2a RET Kinase Protein Amino AcidSubstitutions/Insertions/Deletions^(A) Amino acid position 20 Amino acidposition 32 (e.g., S32L) Amino acid position 34 (e.g., D34S) Amino acidposition 40 (e.g., L40P) Amino acid position 64 (e.g., P64L) Amino acidposition 67 (e.g., R67H) Amino acid position 114 (e.g., R114H) Aminoacid position 145 (e.g., V145G) Amino acid position 200 Amino acidposition 292 (e.g., V292M) Amino acid position 294 Amino acid position321 (e.g., G321R) Amino acid position 330 (e.g., R330Q) Amino acidposition 338 (e.g., T338I) Amino acid position 360 (e.g., R360W) Aminoacid position 393 (e.g., F393L) Amino acid position 432 Δ Amino acidresidues 505-506 (6-Base Pair In-Frame Germline Deletion in Exon 7)Amino acid position 510 (e.g., A510V) Amino acid position 511 (e.g.,E511K) Amino acid position 513 (e.g., G513D) Amino acid position 515(e.g., C515S, C515W⁴) Amino acid position 525 (e.g., R525W) Amino acidposition 531 (e.g., C531R, or 9 base pair duplication) Amino acidposition 532 (e.g., duplication) Amino acid position 533 (e.g., G533C,G533S) Amino acid position 550 (e.g., G550E) Amino acid position 591(e.g., V591I) Amino acid position 593 (e.g., G593E) Amino acid position595 (e.g., E595D and E595A) Amino acid position 600 (e.g., R600Q) Aminoacid position 602 (e.g., I602V) Amino acid position 603 (e.g., K603Q,K603E) Amino acid position 606 (e.g., Y606C) Amino acid position 609(e.g., C609Y, C609S, C609G, C609R, C609F, C609W) Amino acid position 611(e.g., C611R, C611S, C611G, C611Y, C611F, C611W) Amino acid position 616(e.g., E616Q) Amino acid position 618 (e.g., C618S, C618Y, C618R, C618G,C618F, C618W) Amino acid position 620 (e.g., C620S, C620W, C620R, C620G,C620L, C620Y, C620F) Amino acid position 623 (e.g., E623K) Amino acidposition 624 (e.g., D624N) Amino acid position 630 (e.g., C630A, C630R,C630S, C630Y, C630F, C630W) Amino acid position 631 (e.g., D631N, D631Y,D631A, D631G, D631V, D631E,) Amino acid position 632 (e.g., E632K,E632G) Δ Amino acid residues 632-633 (6-Base Pair In-Frame GermlineDeletion in Exon 11) Amino acid position 633 (e.g., 9 base pairduplication) Amino acid position 634 (e.g., C634W, C634Y, C634S, C634R,C634F, C634G, C634L, C634A, or C634T, or an insertion ELCR, or a 12 basepair duplication) (e.g., causing MTC) Amino acid position 635 (e.g.,R635G) Amino acid position 636 (e.g., T636P, T636M) Amino acid position640 (e.g., A640G) Amino acid position 641 (e.g., A641S, A641T) Aminoacid position 648 (e.g., V648I) Amino acid position 649 (e.g., S649L)Amino acid position 664 (e.g., A664D) Amino acid position 665 (e.g.,H665Q) Amino acid position 666 (e.g., K666E, K666M, K666N, K666R) Aminoacid position 686 (e.g., S686N) Amino acid position 689 (e.g., S689T)Amino acid position 691 (e.g., G691S) Amino acid position 694 (e.g.,R694Q) Amino acid position 700 (e.g., M700L) Amino acid position 706(e.g., V706M, V706A) Amino acid position 713 splice variant (e.g.,E713K) Amino acid position 732 (e.g., E732K) Amino acid position 736(e.g., G736R) Amino acid position 748 (e.g., G748C) Amino acid position750 (e.g., A750P) Amino acid position 765 (e.g., S765P) Amino acidposition 766 (e.g., P766S, P766M) Amino acid position 768 (e.g., E768Q,E768D) Amino acid position 769 (e.g., L769L) Amino acid position 770(e.g., R770Q) Amino acid position 771 (e.g., D771N) Amino acid position777 (e.g., N777S) Amino acid position 778 (e.g., V778I) Amino acidposition 781 (e.g., Q781R) Amino acid position 790 (e.g., L790F) Aminoacid position 791 (e.g., Y791F, Y791N) Amino acid position 802 Aminoacid position 804 (e.g., V804L, V804M, V804E) (e.g., causing MTC) Aminoacid position 805 (e.g., E805K) Amino acid position 804/805 (e.g.,V804M/E805K) Amino acid position 806 (e.g., Y806F, Y806S, Y806G, Y806C,Y806E, Y806H, Y806N) Amino acid position 810 (e.g., G810R, G810S, G810A,G810C, G810V, and G810D) Amino acid position 818 (e.g., E818K) Aminoacid position 819 (e.g., S819I) Amino acid position 823 (e.g., G823E)Amino acid position 826 (e.g., Y826M, Y826S) Amino acid position 833(e.g., R833C) Amino acid position 836 (e.g., S836S) Amino acid position841 (e.g., P841L, P841P) Amino acid position 843 (e.g., E843D) Aminoacid position 844 (e.g., R844W, R844Q, R844L) Amino acid position 848(e.g., M848T) Amino acid position 852 (e.g., I852M) Amino acid position865 (e.g., L865V) Amino acid position 870 (e.g., L870F) Amino acidposition 873 (e.g., R873W) Amino acid position 876 (e.g., A876V) Aminoacid position 881 (e.g., L881V) Amino acid position 882 Amino acidposition 883 (e.g., A883F, A883S, A883T) Amino acid position 884 (e.g.,E884K) Amino acid position 886 (e.g., R886W) Amino acid position 891(e.g., S891A) Amino acid position 897 (e.g., R897Q) Amino acid position898 (e.g., D898V) Amino acid position 900 (e.g., Y900F) Amino acidposition 901 (e.g., E901K) Amino acid position 904 (e.g., S904F, S904S,S904C) Amino acid position 907 (e.g., K907E, K907M) Amino acid position908 (e.g., R908K) Amino acid position 911 (e.g., G911D) Amino acidposition 912 (e.g., R912P, R912Q) Amino acid position 918 (e.g., M918T,M918V, M918L) (e.g., causing MTC) Amino acid position 919 (e.g., A919V)Amino acid position 921 (e.g., E921K) Amino acid position 922 (e.g.,S922P, S922Y) Amino acid position 930 (e.g., T930M) Amino acid position961 (e.g., F961L) Amino acid position 972 (e.g., R972G) Amino acidposition 982 (e.g., R982C) Amino acid position 1009 (e.g., M1009V) Aminoacid position 1015 (e.g., Y1015F) Amino acid position 1017 (e.g.,D1017N) Amino acid position 1041 (e.g., V1041G) Amino acid position 1064(e.g., M1064T) Amino acid position 1096 (e.g., Y1096F) RET + 3 (In-FrameDeletion in Exons 6 and 11) (3bp In-Frame Deletion in Exon 15) ^(A)TheRET kinase mutations shown above may be activating mutations and/or mayconfer increased resistance of the RET kinase to a RET inhibitor and/ora multi-kinase inhibitor (MKI), e.g., as compared to a wildtype RETkinase.

In some embodiments, the dysregulation of a RET gene, a RET kinase, orexpression or activity or level of any of the same, includes a splicevariation in a RET mRNA which results in an expressed protein that is analternatively spliced variant of RET having at least one residue deleted(as compared to the wild-type RET kinase) resulting in a constitutiveactivity of a RET kinase domain.

A “RET kinase inhibitor” as defined herein includes any compoundexhibiting RET inhibition activity. In some embodiments, a RET kinaseinhibitor is selective for a RET kinase. Exemplary RET kinase inhibitorscan exhibit inhibition activity (IC₅₀) against a RET kinase of less thanabout 1000 nM, less than about 500 nM, less than about 200 nM, less thanabout 100 nM, less than about 50 nM, less than about 25 nM, less thanabout 10 nM, or less than about 1 nM as measured in an assay asdescribed herein. In some embodiments, a RET kinase inhibitor canexhibit inhibition activity (IC₅₀) against a RET kinase of less thanabout 25 nM, less than about 10 nM, less than about 5 nM, or less thanabout 1 nM as measured in an assay as provided herein.

As used herein, a “first RET kinase inhibitor” or “first RET inhibitor”is a RET kinase inhibitor as defined herein, but which does not includea compound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof as defined herein. As used herein,a “second RET kinase inhibitor” or a “second RET inhibitor” is a RETkinase inhibitor as defined herein, but which does not include acompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof as defined herein. When both afirst and a second RET inhibitor are present in a method providedherein, the first and second RET kinase inhibitor are different.

In some embodiments, the dysregulation of a RET gene, a RET kinase, orexpression or activity or level of any of the same, includes at leastone point mutation in a RET gene that results in the production of a RETkinase that has one or more amino acid substitutions or insertions ordeletions in a RET gene that results in the production of a RET kinasethat has one or more amino acids inserted or removed, as compared to thewild-type RET kinase. In some cases, the resulting RET kinase is moreresistant to inhibition of its phosphotransferase activity by one ormore first RET kinase inhibitor(s), as compared to a wildtype RET kinaseor a RET kinase not including the same mutation. Such mutations,optionally, do not decrease the sensitivity of the cancer cell or tumorhaving the RET kinase to treatment with a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof(e.g., as compared to a cancer cell or a tumor that does not include theparticular RET inhibitor resistance mutation). In such embodiments, aRET inhibitor resistance mutation can result in a RET kinase that hasone or more of an increased V_(max), a decreased K_(m) for ATP, and anincreased K_(D) for a first RET kinase inhibitor, when in the presenceof a first RET kinase inhibitor, as compared to a wildtype RET kinase ora RET kinase not having the same mutation in the presence of the samefirst RET kinase inhibitor.

In other embodiments, the dysregulation of a RET gene, a RET kinase, orexpression or activity or level of any of the same, includes at leastone point mutation in a RET gene that results in the production of a RETkinase that has one or more amino acid substitutions as compared to thewild-type RET kinase, and which has increased resistance to a compoundof Formula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof, as compared to a wildtype RET kinase or a RETkinase not including the same mutation. In such embodiments, a RETinhibitor resistance mutation can result in a RET kinase that has one ormore of an increased V_(max), a decreased K_(m), and a decreased K_(D)in the presence of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof, as compared to awildtype RET kinase or a RET kinase not having the same mutation in thepresence of the same compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof.

Examples of RET inhibitor resistance mutations can, e.g., include pointmutations, insertions, or deletions in and near the ATP binding site inthe tertiary structure of RET kinase (e.g., amino acid positions730-733, 738, 756, 758, 804, 805, 807, 810, 811, 881, and 892 of awildtype RET kinase, e.g., the exemplary wildtype RET kinase describedherein), including but not limited to a gatekeeper residue (e.g., aminoacid position 804 in a wildtype RET kinase), P-loop residues (e.g.,amino acid positions 730-737 in a wildtype RET kinase), residues in ornear the DFG motif (e.g., amino acid positions 888-898 in a wildtype RETkinase), and ATP cleft solvent front amino acid residues (e.g., aminoacid positions 758, 811, and 892 of a wildtype RET kinase). Additionalexamples of these types of mutations include changes in residues thatmay affect enzyme activity and/or drug binding including but are notlimited to residues in the activation loop (e.g., amino acid positions891-916 of a wildtype RET kinase), residues near or interacting with theactivation loop, residues contributing to active or inactive enzymeconformations, changes including mutations, deletions, and insertions inthe loop proceeding the C-helix and in the C-helix (e.g., amino acidpositions 768-788 in a wildtype RET protein). In some embodiments, thewildtype RET protein is the exemplary wildtype RET kinase describedherein. Specific residues or residue regions that may be changed (andare RET inhibitor resistance mutations) include but are not limited tothose listed in Table 3, with numbering based on the human wildtype RETprotein sequence (e.g., SEQ ID NO: 1). As can be appreciated by thoseskilled in the art, an amino acid position in a reference proteinsequence that corresponds to a specific amino acid position in SEQ IDNO: 1 can be determined by aligning the reference protein sequence withSEQ ID NO: 1 (e.g., using a software program, such as ClustalW2).Additional examples of RET inhibitor resistance mutation positions areshown in Table 4. Changes to these residues may include single ormultiple amino acid changes, insertions within or flanking thesequences, and deletions within or flanking the sequences. See also J.Kooistra, G. K. Kanev, O. P. J. Van Linden, R. Leurs, I. J. P. De Esch,and C. De Graaf, “KLIFS: A structural kinase-ligand interactiondatabase,” Nucleic Acids Res., vol. 44, no. D1, pp. D365-D371, 2016,which is incorporated by reference in its entirety herein.

Exemplary Sequence of Mature Human RET Protein (SEQ ID NO: 1)MAKATSGAAG LRLLLLLLLP LLGKVALGLY FSRDAYWEKLYVDQAAGTPL LYVHALRDAP EEVPSFRLGQ HLYGTYRTRLHENNWICIQE DTGLLYLNRS LDHSSWEKLS VRNRGFPLLTVYLKVFLSPT SLREGECQWP GCARVYFSFF NTSFPACSSLKPRELCFPET RPSFRIRENR PPGTFHQFRL LPVQFLCPNISVAYRLLEGE GLPFRCAPDS LEVSTRWALD REQREKYELVAVCTVHAGAR EEVVMVPFPV TVYDEDDSAP TFPAGVDTASAVVEFKRKED TVVATLRVFD ADVVPASGEL VRRYTSTLLP GDTWAQQTFR VEHWPNETSV QANGSFVRAT VHDYRLVLNRNLSISENRTM QLAVLVNDSD FQGPGAGVLL LHFNVSVLPVSLHLPSTYSL SVSRRARRFA QIGKVCVENC QAFSGINVQYKLHSSGANCS TLGVVTSAED TSGILFVNDT KALRRPKCAELHYMVVATDQ QTSRQAQAQL LVTVEGSYVA EEAGCPLSCAVSKRRLECEE CGGLGSPTGR CEWRQGDGKG ITRNFSTCSPSTKTCPDGHC DVVETQDINI CPQDCLRGSI VGGHEPGEPR GIKAGYGTCN CFPEEEKCFC EPEDIQDPLC DELCRTVIAAAVLFSFIVSV LLSAFCIHCY HKFAHKPPIS SAEMTFRRPAQAFPVSYSSS GARRPSLDSM ENQVSVDAFK ILEDPKWEFPRKNLVLGKTL GEGEFGKVVK ATAFHLKGRA GYTTVAVKMLKENASPSELR DLLSEFNVLK QVNHPHVIKL YGACSQDGPLLLIVEYAKYG SLRGFLRESR KVGPGYLGSG GSRNSSSLDHPDERALTMGD LISFAWQISQ GMQYLAEMKL VHRDLAARNILVAEGRKMKI SDFGLSRDVY EEDSYVKRSQ GRIPVKWMAIESLFDHIYTT QSDVWSFGVL LWEIVTLGGN PYPGIPPERLFNLLKTGHRM ERPDNCSEEM YRLMLQCWKQ EPDKRPVFADISKDLEKMMV KRRDYLDLAA STPSDSLIYD DGLSEEETPLVDCNNAPLPR ALPSTWIENK LYGMSDPNWP GESPVPLTRADGTNTGFPRY PNDSVYANWM LSPSAAKLMD TFDS

In some embodiments, a RET inhibitor resistance mutation can include adysregulation of a MET gene, a MET kinase, or the expression or activityor level of any of the same.

The phrase “dysregulation of a MET gene, a MET kinase, or the expressionor activity or level of any of the same” refers to a genetic mutation(e.g., a MET gene translocation that results in the expression of afusion protein, a deletion in a MET gene that results in the expressionof a RET protein that includes a deletion of at least one amino acid ascompared to the wild-type RET protein, or a mutation in a MET gene thatresults in the expression of a RET protein with one or more pointmutations, or an alternative spliced version of a MET mRNA that resultsin a MET protein that results in the deletion of at least one amino acidin the MET protein as compared to the wild-type MET protein), or a METgene amplification that results in overexpression of a MET protein or anautocrine activity resulting from the overexpression of a MET gene acell, that results in a pathogenic increase in the activity of a kinasedomain of a MET protein (e.g., a constitutively active kinase domain ofa MET protein) in a cell. As another example, a dysregulation of a METgene, a MET protein, or expression or activity, or level of any of thesame, can be a mutation in a MET gene that encodes a MET protein that isconstitutively active or has increased activity as compared to a proteinencoded by a MET gene that does not include the mutation. For example, adysregulation of a MET gene, a MET protein, or expression or activity,or level of any of the same, can be the result of a gene or chromosometranslocation which results in the expression of a fusion protein thatcontains a first portion of MET that includes a functional kinasedomain, and a second portion of a partner protein (i.e., that is notMET). In some examples, dysregulation of a MET gene, a MET protein, orexpression or activity, can be a result of a gene translocation of oneMET gene with another non-MET gene.

The term “wildtype MET” or “wild-type MET” describes a nucleic acid(e.g., a MET gene or a MET mRNA) or protein (e.g., a MET protein) thatis found in a subject that does not have a MET-associated cancer (andoptionally also does not have an increased risk of developing aMET-associated cancer and/or is not suspected of having a MET-associatedcancer), or is found in a cell or tissue from a subject that does nothave a MET-associated cancer (and optionally also does not have anincreased risk of developing a MET-associated cancer and/or is notsuspected of having a MET-associated cancer). The term “MET-associatedcancer” as used herein refers to cancers associated with or having adysregulation of a MET gene, a MET kinase, or expression or activity, orlevel of any of the same.

In some embodiments, compounds of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof are useful intreating patients that develop cancers with RET inhibitor resistancemutations (e.g., that result in an increased resistance to a first RETinhibitor, e.g., a substitution at amino acid position 804, e.g., V804M,V804L, or V804E, a substitution at amino acid position 810, e.g., G810S,G810R, G810C, G810A, G810V, and G810D, and/or one or more RET inhibitorresistance mutations listed in Tables 3 and 4) by either dosing incombination or as a subsequent or additional (e.g., follow-up) therapyto existing drug treatments (e.g., other RET kinase inhibitors; e.g.,first and/or second RET kinase inhibitors). Exemplary first and secondRET kinase inhibitors are described herein. In some embodiments, a firstor second RET kinase inhibitor can be selected from the group consistingof cabozantinib, vandetanib, alectinib, apatinib, sitravatinib,sorafenib, lenvatinib, ponatinib, dovitinib, sunitinib, foretinib,BLU667, and BLU6864.

In some embodiments, compounds of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof are useful fortreating a cancer that has been identified as having one or more RETinhibitor resistance mutations (that result in an increased resistanceto a first or second RET inhibitor, e.g., a substitution at amino acidposition 804, e.g., V804M, V804L, or V804E, or e.g., a substitution atamino acid position 810, e.g., G810S, G810R, G810C, G810A, G810V, andG810D). In some embodiments, the one or more RET inhibitor resistancemutations occur in a nucleic acid sequence encoding a RET fusion protein(e.g. any of the RET gene fusion proteins described in Table 1)resulting in a RET fusion protein that exhibits RET kinase inhibitorresistance. In some embodiments, the one or more RET inhibitorresistance mutations occurs in a nucleic acid sequence encoding a mutantRET protein (e.g. a mutant RET protein having any of the mutationsdescribed in Table 2) resulting in a mutant RET protein that exhibitsRET kinase resistance. Non-limiting examples of RET inhibitor resistancemutations are listed in Tables 3 and 4.

TABLE 3 RET Inhibitor Resistance Mutations Exemplary RET ResistanceMutations Amino acid position 634 (e.g., C634W)¹⁰ Amino acid position732 (e.g., E732K)⁷ Amino acid position 788 (e.g., I788N)⁸ Amino acidposition 790 (e.g., L790F)⁹ Amino acid position 804 (e.g., V804M^(1,2),V804L^(1,2), V804E⁶) Amino acid position 778/804¹³ Amino acid position804/805 (e.g., V804M/E805K)³ Amino acid position 806 (e.g., Y806C^(4,6),Y806E⁴, Y806S⁶, Y806H⁶, Y806N⁶) Amino acid position 804/806 (e.g.,V804M/Y806C)¹¹ Amino acid position 810 (e.g., G810A⁵, G810R⁶, G810S⁶,G810C, G810V, and G810D) Amino acid position 865 (e.g., L865V⁶) Aminoacid position 870 (e.g., L870F⁶) Amino acid position 891 (e.g., S891A)¹⁰Amino acid position 904 (e.g., S904F)¹² Amino acid position 804/904(e.g., V804M/S904C)¹¹ Amino acid position 918 (e.g.. M918T)¹⁰ ¹Yoon etal., J. Med. Chem. 59(1): 358-73, 2016. ²U.S. Pat. No. 8,629,135.³Cranston, et al., Cancer Res. 66(20): 10179-87, 2006. ⁴Carlomagno, etal., Endocr. Rel. Cancer 16(1): 233-41, 2009. ⁵Huang et al., Mol. CancerTher., 2016 Aug. 5. pii: molcanther.0258.2016. [Epub ahead of print].⁶PCT patent application Pub. No. WO 2016/127074. ⁷Mamedova et al.,Summer Undergraduate Research Programs (SURP) Student Abstracts,University of Oklahoma Health Sciences Center, 2016. ⁸Plenker et al.,Sci. Transl. Med., 9(394), doi: 10.1126/scitranslmed.aah6144, 2017.⁹Kraft et al, Cancer Research, 2017, Vol. 77, No. 13, Supp.Supplement 1. Abstract Number: 4882; American Association for CancerResearch Annual Meeting 2017. Washington, DC, United States. 1 Apr.2017-5 Apr. 2017. ¹⁰U.S. patent application Pub. No. 2018/0022732.¹¹Roskoski and Sadeghi-Nejad, Pharmacol. Res., 128, 1-17. doi:10.1016/j.phrs.2017.12.021, 2018. ¹²Nakaoku, et al. Nat Commun, 9(1),625. doi: 10.1038/s41467-018-02994-7, 2018. ¹³Roy et al. Oncologist,18(10): 1093-1100. doi: 10.1634/theoncologist.2013-0053, 2013.

TABLE 4 Additional Exemplary Amino Acid Positions of RET InhibitorResistance Mutations RET Amino Acid and Exemplary Position MutationMechanistic Resistance Rationale L730 P Steric hindrance and/or activeconformational effect G731 V Steric hindrance and/or activeconformational effect E732 K Steric hindrance and/or activeconformational effect G733 V Steric hindrance and/or activeconformational effect E734 K Steric hindrance and/or activeconformational effect L760 M Active conformational effect K761 E Activeconformational effect E762 K Active conformational effect N763 D Activeconformational effect A764 V Active conformational effect S765 N Activeconformational effect P766 A Active conformational effect S767 C Activeconformational effect E768 K Active conformational effect L779 M Sterichindrance and/or active conformational effect I788 M Steric hindranceand/or active conformational effect M868 R Steric hindrance and/oractive conformational effect K869 E Steric hindrance and/or activeconformational effect L870 Q Steric hindrance and/or activeconformational effect V871 M Steric hindrance and/or activeconformational effect H872 R Steric hindrance and/or activeconformational effect R873 P Steric hindrance and/or activeconformational effect D874 Y Steric hindrance and/or activeconformational effect L881 R Steric hindrance and/or activeconformational effect L895 M Active conformational effect S896 N Activeconformational effect R897 C Active conformational effect D898 Y Activeconformational effect V899 G Active conformational effect Y900 D Activeconformational effect E901 K Active conformational effect E902 K Activeconformational effect D903 Y Active conformational effect S904 C Activeconformational effect Y905 D Active conformational effect V906 M Activeconformational effect K907 E Active conformational effect R908 P Activeconformational effect S909 C Active conformational effect Q910 R Activeconformational effect G911 C Active conformational effect R912 P Activeconformational effect

The oncogenic role of RET was first described in papillary thyroidcarcinoma (PTC) (Grieco et al., Cell, 1990, 60, 557-63), which arisesfrom follicular thyroid cells and is the most common thyroid malignancy.Approximately 20-30% of PTC harbor somatic chromosomal rearrangements(translocations or inversions) linking the promoter and the 5′ portionsof constitutively expressed, unrelated genes to the RET tyrosine kinasedomain (Greco et al., Q. J. Nucl. Med. Mol. Imaging, 2009, 53, 440-54),therefore driving its ectopic expression in thyroid cells. Fusionproteins generated by such rearrangements are termed “RET/PTC” proteins.For example, RET/PTC 1 is a fusion between CCDD6 and RET that iscommonly found in papillary thyroid carcinomas. Similarly, both RET/PTC3and RET/PTC4 are fusions of ELE1 and RET that are commonly found inpapillary thyroid carcinomas, although the fusion events resultingRET/PTC3 and RET/PTC4 lead to different proteins with differentmolecular weights (see e.g., Fugazzola et al., Oncogene, 13(5):1093-7,1996). Some RET fusions associated with PTC are not referred to as“RET/PTC”, but instead are referred to as the fusion protein inself. Forexample, fusion between RET and both ELKS and PCM1 are found in PTCs,but the fusion proteins are referred to as ELKS-RET and PCM1-RET (seee.g., Romei and Elisei, Front. Endocrinol. (Lausanne), 3:54, doi:10.3389/fendo.2012.00054, 2012). The role of RET-PTC rearrangements inthe pathogenesis of PTC has been confirmed in transgenic mice (Santoroet al., Oncogene, 1996, 12, 1821-6). To date, a variety of fusionpartners have been identified, from PTC and other cancer types, allproviding a protein/protein interaction domain that inducesligand-independent RET dimerization and constitutive kinase activity(see, e.g., Table 1). Recently, a 10.6 Mb pericentric inversion inchromosome 10, where RET gene maps, has been identified in about 2% oflung adenocarcinoma patients, generating different variants of thechimeric gene KIF5B-RET (Ju et al., Genome Res., 2012, 22, 436-45; Kohnoet al., 2012, Nature Med., 18, 375-7; Takeuchi et al., Nature Med.,2012, 18, 378-81; Lipson et al., 2012, Nature Med., 18, 382-4). Thefusion transcripts are highly expressed and all the resulting chimericproteins contain the N-terminal portion of the coiled-coil region ofKIF5B, which mediates homodimerization, and the entire RET kinasedomain. None of RET positive patients harbor other known oncogenicalterations (such as EGFR or K-Ras mutation, ALK translocation),supporting the possibility that KIF5B-RET fusion could be a drivermutation of lung adenocarcinoma. The oncogenic potential of KIF5B-REThas been confirmed by transfecting the fusion gene into cultured celllines: similarly to what has been observed with RET-PTC fusion proteins,KIF5B-RET is constitutively phosphorylated and induces NIH-3T3transformation and IL-3 independent growth of BA-F3 cells. However,other RET fusion proteins have been identified in lung adenocarcinomapatients, such as the CCDC6-RET fusion protein, which has been found toplay a key role in the proliferation of the human lung adenocarcinomacell line LC-2/ad (Journal of Thoracic Oncology, 2012, 7(12):1872-1876).RET inhibitors have been shown to be useful in treating lung cancersinvolving RET rearrangements (Drilon, A. E. et al. J Clin Oncol 33, 2015(suppl; abstr 8007)). RET fusion proteins have also been identified inpatients having colorectal cancer (Song Eun-Kee, et al. InternationalJournal of Cancer, 2015, 136: 1967-1975).

Besides rearrangements of the RET sequence, gain of function pointmutations of RET proto-oncogene are also driving oncogenic events, asshown in medullary thyroid carcinoma (MTC), which arises fromparafollicular calcitonin-producing cells (de Groot, et al., EndocrineRev., 2006, 27, 535-60; Wells and Santoro, Clin. Cancer Res., 2009, 15,7119-7122). Around 25% of MTC are associated with multiple endocrineneoplasia type 2 (MEN2), a group of inherited cancer syndromes affectingneuroendocrine organs caused by germline activating point mutations ofRET. In MEN2 subtypes (MEN2A, MEN2B and Familial MTC/FMTC) RET genemutations have a strong phenotype-genotype correlation definingdifferent MTC aggressiveness and clinical manifestations of the disease.In MEN2A syndrome mutations involve one of the six cysteine residues(mainly C634) located in the cysteine-rich extracellular region, leadingto ligand-independent homodimerization and constitutive RET activation.Patients develop MTC at a young age (onset at 5-25 years) and may alsodevelop pheochromocytoma (50%) and hyperparathyroidism. MEN2B is mainlycaused by M918T mutation, which is located in the kinase domain. Thismutation constitutively activates RET in its monomeric state and alterssubstrate recognition by the kinase. MEN2B syndrome is characterized byan early onset (<1 year) and very aggressive form of MTC,pheochromocytoma (50% of patients) and ganglioneuromas. In FMTC the onlydisease manifestation is MTC, usually occurring at an adult age. Manydifferent mutations have been detected, spanning the entire RET gene.The remaining 75% of MTC cases are sporadic and about 50% of them harborRET somatic mutations: the most frequent mutation is M918T that, as inMEN2B, is associated with the most aggressive phenotype. Somatic pointmutations of RET have also been described in other tumors such ascolorectal cancer (Wood et al., Science, 2007, 318, 1108-13) and smallcell lung carcinoma (Jpn. J Cancer Res., 1995, 86, 1127-30). In someembodiments, the MTC is RET-fusion positive MTC.

RET signaling components have been found to be expressed in primarybreast tumors and to functionally interact with estrogen receptor-ccpathway in breast tumor cell lines (Boulay et al., Cancer Res. 2008, 68,3743-51; Plaza-Menacho et al., Oncogene, 2010, 29, 4648-57), while RETexpression and activation by GDNF family ligands could play an importantrole in perineural invasion by different types of cancer cells (Ito etal., Surgery, 2005, 138, 788-94; Gil et al., J. Natl. Cancer Inst.,2010, 102, 107-18; Iwahashi et al., Cancer, 2002, 94, 167-74).

RET is also expressed in 30-70% of invasive breast cancers, withexpression being relatively more frequent in estrogen receptor-positivetumors (Plaza-Menacho, I., et al., Oncogene, 2010, 29, 4648-4657;Esseghir, S., et al., Cancer Res., 2007, 67, 11732-11741; Morandi, A.,et al., Cancer Res., 2013, 73, 3783-3795; Gattelli, A., EMBO Mol. Med.,2013, 5, 1335-1350).

The identification of RET rearrangements has been reported in a subsetof (patient-derived xenograft) PDX established from colorectal cancer.Although the frequency of such events in colorectal cancer patientsremains to be defined, these data suggest a role of RET as a target inthis indication (Gozgit et al., AACR Annual Meeting 2014). Studies haveshown that the RET promoter is frequently methylated in colorectalcancers, and heterozygous missense mutations, which are predicted toreduce RET expression, are identified in 5-10% of cases, which suggeststhat RET might have some features of a tumor suppressor in sporadiccolon cancers (Luo, Y., et al., Oncogene, 2013, 32, 2037-2047; Sjoblom,T., et al., Science, 2006, 268-274; Cancer Genome Atlas Network, Nature,2012, 487, 330-337).

An increasing number of tumor types are now being shown to expresssubstantial levels of wild-type RET kinase that could have implicationsfor tumor progression and spread. RET is expressed in 50-65% ofpancreatic ductal carcinomas, and expression is more frequent inmetastatic and higher grade tumors (Ito, Y, et al., Surgery, 2005, 138,788-794; Zeng, Q., et al., J. Int. Med. Res. 2008, 36, 656-664).

In neoplasms of hematopoietic lineages, RET is expressed in acutemyeloid leukemia (AML) with monocytic differentiation, as well as inCMML (Gattei, V. et al., Blood, 1997, 89, 2925-2937; Gattei, V., et al.,Ann. Hematol, 1998, 77, 207-210; Camos, M., Cancer Res. 2006, 66,6947-6954). Recent studies have identified rare chromosomalrearrangements that involve RET in patients with chronic myelomonocyticleukemia (CMML). CMML is frequently associated with rearrangements ofseveral tyrosine kinases, which result in the expression of chimericcytosolic oncoproteins that lead to activation of RAS pathways(Kohlmann, A., et al., J. Clin. Oncol. 2010, 28, 2858-2865). In the caseof RET, gene fusions that link RET with BCR (BCR-RET) or with fibroblastgrowth factor receptor 1 oncogene partner (FGFR1OP-RET) weretransforming in early hematopoietic progenitor cells and could shiftmaturation of these cells towards monocytic paths, probably through theinitiation of RET-mediated RAS signaling (Ballerini, P., et al.,Leukemia, 2012, 26, 2384-2389).

RET expression has also been shown to occur in several other tumortypes, including prostate cancer, small-cell lung carcinoma, melanoma,renal cell carcinoma, and head and neck tumors (Narita, N., et al.,Oncogene, 2009, 28, 3058-3068; Mulligan, L. M., et al., GenesChromosomes Cancer, 1998, 21, 326-332; Flavin, R., et al., Urol. Oncol.,2012, 30, 900-905; Dawson, D. M., J Natl Cancer Inst, 1998, 90,519-523).

In neuroblastoma, RET expression and activation by GFLs has roles intumor cell differentiation, potentially collaborating with otherneurotrophic factor receptors to down regulate N-Myc, the expression ofwhich is a marker of poor prognosis (Hofstra, R. M., W., et al., Hum.Genet. 1996, 97, 362-364; Petersen, S. and Bogenmann, E., Oncogene,2004, 23, 213-225; Brodeur, G. M., Nature Ref. Cancer, 2003, 3,203-216).

Multitargeted inhibitors which cross react with RET are known (Borrello,M. G., et al., Expert Opin. Ther. Targets, 2013, 17(4), 403-419;International Patent Application Nos. WO 2014/141187, WO 2014/184069,and WO 2015/079251). Such multitargeted inhibitors (or multikinaseinhibitors or MKIs) can also be associated with development of RETinhibitor resistance mutations. See, for example, Q. Huang et al.,“Preclinical Modeling of KIF5B-RET Fusion Lung Adenocarcinoma.,” Mol.Cancer Ther., no. 18, pp. 2521-2529, 2016; Yasuyuki Kaneta et al.,Abstract B173: Preclinical characterization and antitumor efficacy ofDS-5010, a highly potent and selective RET inhibitor, Mol Cancer TherJan. 1 2018 (17) (1 Supplement) B173;DOI:10.1158/1535-7163.TARG-17-B173, both of which are incorporated byreference in their entirety herein.

Accordingly, provided herein are methods for treating a patientdiagnosed with (or identified as having) a cancer that includeadministering to the patient a therapeutically effective amount of acompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof. Also provided herein are methodsfor treating a patient identified or diagnosed as having aRET-associated cancer that include administering to the patient atherapeutically effective amount of a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofor a pharmaceutical composition thereof. In some embodiments, thepatient that has been identified or diagnosed as having a RET-associatedcancer through the use of a regulatory agency-approved, e.g.,FDA-approved test or assay for identifying dysregulation of a RET gene,a RET kinase, or expression or activity or level of any of the same, ina patient or a biopsy sample from the patient or by performing any ofthe non-limiting examples of assays described herein. In someembodiments, the test or assay is provided as a kit. In someembodiments, the cancer is a RET-associated cancer. For example, theRET-associated cancer can be a cancer that includes one or more RETinhibitor resistance mutations.

Also provided are methods for treating cancer in a patient in needthereof, the method comprising: (a) detecting a RET-associated cancer inthe patient; and (b) administering to the patient a therapeuticallyeffective amount of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof or apharmaceutical composition thereof. Some embodiments of these methodsfurther include administering to the subject another anticancer agent(e.g., a second RET inhibitor, a second compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,or an immunotherapy). In some embodiments, the subject was previouslytreated with a first RET inhibitor or previously treated with anotheranticancer treatment, e.g., at least partial resection of the tumor orradiation therapy. In some embodiments, the patient is determined tohave a RET-associated cancer through the use of a regulatoryagency-approved, e.g., FDA-approved test or assay for identifyingdysregulation of a RET gene, a RET kinase, or expression or activity orlevel of any of the same, in a patient or a biopsy sample from thepatient or by performing any of the non-limiting examples of assaysdescribed herein. In some embodiments, the test or assay is provided asa kit. In some embodiments, the cancer is a RET-associated cancer. Forexample, the RET-associated cancer can be a cancer that includes one ormore RET inhibitor resistance mutations.

Also provided are methods of treating a patient that include performingan assay on a sample obtained from the patient to determine whether thepatient has a dysregulation of a RET gene, a RET kinase, or expressionor activity or level of any of the same, and administering (e.g.,specifically or selectively administering) a therapeutically effectiveamount of a compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof or a pharmaceuticalcomposition thereof to the patient determined to have a dysregulation ofa RET gene, a RET kinase, or expression or activity or level of any ofthe same. Some embodiments of these methods further includeadministering to the subject another anticancer agent (e.g., a secondRET inhibitor, a second compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof, orimmunotherapy). In some embodiments of these methods, the subject waspreviously treated with a first RET inhibitor or previously treated withanother anticancer treatment, e.g., at least partial resection of atumor or radiation therapy. In some embodiments, the patient is apatient suspected of having a RET-associated cancer, a patientpresenting with one or more symptoms of a RET-associated cancer, or apatient having an elevated risk of developing a RET-associated cancer.In some embodiments, the assay utilizes next generation sequencing,pyrosequencing, immunohistochemistry, or break apart FISH analysis. Insome embodiments, the assay is a regulatory agency-approved assay, e.g.,FDA-approved kit. In some embodiments, the assay is a liquid biopsy.Additional, non-limiting assays that may be used in these methods aredescribed herein. Additional assays are also known in the art. In someembodiments, the dysregulation of a RET gene, a RET kinase, orexpression or activity or level of any of the same includes one or moreRET inhibitor resistance mutations.

Also provided is a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof or apharmaceutical composition thereof for use in treating a RET-associatedcancer in a patient identified or diagnosed as having a RET-associatedcancer through a step of performing an assay (e.g., an in vitro assay)on a sample obtained from the patient to determine whether the patienthas a dysregulation of a RET gene, a RET kinase, or expression oractivity or level of any of the same, where the presence of adysregulation of a RET gene, a RET kinase, or expression or activity orlevel of any of the same, identifies that the patient has aRET-associated cancer. Also provided is the use of a compound of FormulaI-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphform thereof for the manufacture of a medicament for treating aRET-associated cancer in a patient identified or diagnosed as having aRET-associated cancer through a step of performing an assay on a sampleobtained from the patient to determine whether the patient has adysregulation of a RET gene, a RET kinase, or expression or activity orlevel of any of the same where the presence of dysregulation of a RETgene, a RET kinase, or expression or activity or level of any of thesame, identifies that the patient has a RET-associated cancer. Someembodiments of any of the methods or uses described herein furtherinclude recording in the patient's clinical record (e.g., a computerreadable medium) that the patient is determined to have a dysregulationof a RET gene, a RET kinase, or expression or activity or level of anyof the same, through the performance of the assay, should beadministered a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof or apharmaceutical composition thereof. In some embodiments, the assayutilizes next generation sequencing, pyrosequencing,immunohistochemistry, or break apart FISH analysis. In some embodiments,the assay is a regulatory agency-approved assay, e.g., FDA-approved kit.In some embodiments, the assay is a liquid biopsy. In some embodiments,the dysregulation of a RET gene, a RET kinase, or expression or activityor level of any of the same includes one or more RET inhibitorresistance mutations.

Also provided is a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof, for use in thetreatment of a cancer in a patient in need thereof or a patientidentified or diagnosed as having a RET-associated cancer. Also providedis the use of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof for themanufacture of a medicament for treating a cancer in a patientidentified or diagnosed as having a RET-associated cancer. In someembodiments, the cancer is a RET-associated cancer, for example, aRET-associated cancer having one or more RET inhibitor resistancemutations. In some embodiments, a patient is identified or diagnosed ashaving a RET-associated cancer through the use of a regulatoryagency-approved, e.g., FDA-approved, kit for identifying dysregulationof a RET gene, a RET kinase, or expression or activity or level of anyof the same, in a patient or a biopsy sample from the sample. Asprovided herein, a RET-associated cancer includes those described hereinand known in the art.

In some embodiments of any of the methods or uses described herein, thepatient has been identified or diagnosed as having a cancer with adysregulation of a RET gene, a RET kinase, or expression or activity orlevel of any of the same. In some embodiments of any of the methods oruses described herein, the patient has a tumor that is positive for adysregulation of a RET gene, a RET kinase, or expression or activity orlevel of any of the same. In some embodiments of any of the methods oruses described herein, the patient can be a patient with a tumor(s) thatis positive for a dysregulation of a RET gene, a RET kinase, orexpression or activity or level of any of the same. In some embodimentsof any of the methods or uses described herein, the patient can be apatient whose tumors have a dysregulation of a RET gene, a RET kinase,or expression or activity or level of any of the same. In someembodiments of any of the methods or uses described herein, the patientis suspected of having a RET-associated cancer (e.g., a cancer havingone or more RET inhibitor resistance mutations). In some embodiments,provided herein are methods for treating a RET-associated cancer in apatient in need of such treatment, the method comprising a) detecting adysregulation of a RET gene, a RET kinase, or the expression or activityor level of any of the same in a sample from the patient; and b)administering a therapeutically effective amount of a compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof. In some embodiments, the dysregulation of a RETgene, a RET kinase, or the expression or activity or level of any of thesame includes one or more fusion proteins. Non-limiting examples of RETgene fusion proteins are described in Table 1. In some embodiments, thefusion protein is KIF5B-RET. In some embodiments, the dysregulation of aRET gene, a RET kinase, or the expression or activity or level of any ofthe same includes one or more RET kinase protein pointmutations/insertions/deletions. Non-limiting examples of RET kinaseprotein point mutations/insertions/deletions are described in Tables 2and 2a. In some embodiments, the RET kinase protein pointmutations/insertions/deletions are selected from the group consisting ofM918T, M918V, C634W, V804L, V804M, G810S, and G810R. In someembodiments, the dysregulation of a RET gene, a RET kinase, or theexpression or activity or level of any of the same includes one or moreRET inhibitor resistance mutations. Non-limiting examples of RETinhibitor resistance mutations are described in Tables 3 and 4. In someembodiments, the RET inhibitor resistance mutation is V804M. In someembodiments, the RET inhibitor resistance mutation is G810S. In someembodiments, the RET inhibitor resistance mutation is G810R. In someembodiments, the cancer with a dysregulation of a RET gene, a RETkinase, or expression or activity or level of any of the same isdetermined using a regulatory agency-approved, e.g., FDA-approved, assayor kit. In some embodiments, the tumor that is positive for adysregulation of a RET gene, a RET kinase, or expression or activity orlevel of any of the same is a tumor positive for one or more RETinhibitor resistance mutations. In some embodiments, the tumor with adysregulation of a RET gene, a RET kinase, or expression or activity orlevel of any of the same is determined using a regulatoryagency-approved, e.g., FDA-approved, assay or kit.

In some embodiments of any of the methods or uses described herein, thepatient has a clinical record indicating that the patient has a tumorthat has a dysregulation of a RET gene, a RET kinase, or expression oractivity or level of any of the same (e.g., a tumor having one or moreRET inhibitor resistance mutations). In some embodiments, the clinicalrecord indicates that the patient should be treated with one or more ofthe compounds of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof or compositions provided herein. Insome embodiments, the cancer with a dysregulation of a RET gene, a RETkinase, or expression or activity or level of any of the same is acancer having one or more RET inhibitor resistance mutations. In someembodiments, the cancer with a dysregulation of a RET gene, a RETkinase, or expression or activity or level of any of the same isdetermined using a regulatory agency-approved, e.g., FDA-approved, assayor kit. In some embodiments, the tumor that is positive for adysregulation of a RET gene, a RET kinase, or expression or activity orlevel of any of the same is a tumor positive for one or more RETinhibitor resistance mutations. In some embodiments, the tumor with adysregulation of a RET gene, a RET kinase, or expression or activity orlevel of any of the same is determined using a regulatoryagency-approved, e.g., FDA-approved, assay or kit.

Also provided are methods of treating a patient that includeadministering a therapeutically effective amount of a compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof to a patient having a clinical record thatindicates that the patient has a dysregulation of a RET gene, a RETkinase, or expression or activity or level of any of the same. Alsoprovided is the use of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof for themanufacture of a medicament for treating a RET-associated cancer in apatient having a clinical record that indicates that the patient has adysregulation of a RET gene, a RET kinase, or expression or activity orlevel of any of the same. Some embodiments of these methods and uses canfurther include: a step of performing an assay (e.g., an in vitro assay)on a sample obtained from the patient to determine whether the patienthas a dysregulation of a RET gene, a RET kinase, or expression oractivity or level of any of the same, and recording the information in apatient's clinical file (e.g., a computer readable medium) that thepatient has been identified to have a dysregulation of a RET gene, a RETkinase, or expression or activity or level of any of the same. In someembodiments, the assay is an in vitro assay. For example, an assay thatutilizes next generation sequencing, immunohistochemistry, or breakapart FISH analysis. In some embodiments, the assay is a regulatoryagency-approved, e.g., FDA-approved, kit. In some embodiments, the assayis a liquid biopsy. In some embodiments, the dysregulation of a RETgene, RET kinase, or expression or activity or level of any of the sameincludes one or more RET inhibitor resistance mutations.

Also provided herein is a method of treating a subject. In someembodiments, the method includes performing an assay on a sampleobtained from the subject to determine whether the subject has adysregulation of a RET gene, a RET protein, or expression or level ofany of the same. In some such embodiments, the method also includesadministering to a subject determined to have a dysregulation of a RETgene, a RET protein, or expression or activity, or level of any of thesame a therapeutically effective amount of a compound of Formula I-IV,or a pharmaceutically acceptable salt, amorphous, or polymorph formthereof. In some embodiments, the method includes determining that asubject has a dysregulation of a RET gene, a RET protein, or expressionor level of any of the same via an assay performed on a sample obtainedfrom the subject. In such embodiments, the method also includesadministering to a subject a therapeutically effective amount of acompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof. In some embodiments, thedysregulation in a RET gene, a RET kinase protein, or expression oractivity of the same is a gene or chromosome translocation that resultsin the expression of a RET fusion protein (e.g., any of the RET fusionproteins described herein). In some embodiments, the RET fusion can beselected from a KIF5B-RET fusion and a CCDC6-RET fusion. In someembodiments, the dysregulation in a RET gene, a RET kinase protein, orexpression or activity or level of any of the same is one or more pointmutation in the RET gene (e.g., any of the one or more of the RET pointmutations described herein). The one or more point mutations in a RETgene can result, e.g., in the translation of a RET protein having one ormore of the following amino acid substitutions: M918T, M918V, C634W,V804L, V804M, G810S, and G810R. In some embodiments, the dysregulationin a RET gene, a RET kinase protein, or expression or activity or levelof any of the same is one or more RET inhibitor resistance mutations(e.g., any combination of the one or more RET inhibitor resistancemutations described herein). Some embodiments of these methods furtherinclude administering to the subject another anticancer agent (e.g., asecond RET inhibitor a second compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,or immunotherapy).

In some embodiments, the compounds provided herein exhibit brain and/orcentral nervous system (CNS) penetrance. Such compounds are capable ofcrossing the blood brain barrier and inhibiting a RET kinase in thebrain and/or other CNS structures. In some embodiments, the compoundsprovided herein are capable of crossing the blood brain barrier in atherapeutically effective amount. For example, treatment of a patientwith cancer (e.g., a RET-associated cancer such as a RET-associatedbrain or CNS cancer) can include administration (e.g., oraladministration) of the compound to the patient. In some suchembodiments, the compounds provided herein are useful for treating aprimary brain tumor or metastatic brain tumor. For example, thecompounds can be used in the treatment of one or more of gliomas such asglioblastoma (also known as glioblastoma multiforme), astrocytomas,oligodendrogliomas, ependymomas, and mixed gliomas, meningiomas,medulloblastomas, gangliogliomas, schwannomas (neurilemmomas), andcraniopharyngiomas (see, for example, the tumors listed in Louis, D. N.et al. Acta Neuropathol 131(6), 803-820 (June 2016)). In someembodiments, the brain tumor is a primary brain tumor. In someembodiments, the patient has previously been treated with anotheranticancer agent, e.g., another RET inhibitor (e.g., a compound that isnot a compound of General Formula I) or a multi-kinase inhibitor. Insome embodiments, the brain tumor is a metastatic brain tumor. In someembodiments, the patient has previously been treated with anotheranticancer agent, e.g., another RET inhibitor (e.g., a compound that isnot a compound of General Formula I) or a multi-kinase inhibitor.

Also provided are methods (e.g., in vitro methods) of selecting atreatment for a patient identified or diagnosed as having aRET-associated cancer. Some embodiments can further includeadministering the selected treatment to the patient identified ordiagnosed as having a RET-associated cancer. For example, the selectedtreatment can include administration of a therapeutically effectiveamount of a compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof. Some embodiments can furtherinclude a step of performing an assay on a sample obtained from thepatient to determine whether the patient has a dysregulation of a RETgene, a RET kinase, or expression or activity or level of any of thesame, and identifying and diagnosing a patient determined to have adysregulation of a RET gene, a RET kinase, or expression or activity orlevel of any of the same, as having a RET-associated cancer. In someembodiments, the cancer is a RET-associated cancer having one or moreRET inhibitor resistance mutations. In some embodiments, the patient hasbeen identified or diagnosed as having a RET-associated cancer throughthe use of a regulatory agency-approved, e.g., FDA-approved, kit foridentifying dysregulation of a RET gene, a RET kinase, or expression oractivity or level of any of the same, in a patient or a biopsy samplefrom the patient. In some embodiments, the RET-associated cancers is acancer described herein or known in the art. In some embodiments, theassay is an in vitro assay. For example, an assay that utilizes the nextgeneration sequencing, immunohistochemistry, or break apart FISHanalysis. In some embodiments, the assay is a regulatoryagency-approved, e.g., FDA-approved, kit. In some embodiments, the assayis a liquid biopsy.

Also provided herein are methods of selecting a treatment for a patient,wherein the methods include a step of performing an assay on a sampleobtained from the patient to determine whether the patient has adysregulation of a RET gene, a RET kinase, or expression or activity orlevel of any of the same (e.g., one or more RET inhibitor resistancemutations), and identifying or diagnosing a patient determined to have adysregulation of a RET gene, a RET kinase, or expression or activity orlevel of any of the same, as having a RET-associated cancer. Someembodiments further include administering the selected treatment to thepatient identified or diagnosed as having a RET-associated cancer. Forexample, the selected treatment can include administration of atherapeutically effective amount of a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofto the patient identified or diagnosed as having a RET-associatedcancer. In some embodiments, the assay is an in vitro assay. Forexample, an assay that utilizes the next generation sequencing,immunohistochemistry, or break apart FISH analysis. In some embodiments,the assay is a regulatory agency-approved, e.g., FDA-approved, kit. Insome embodiments, the assay is a liquid biopsy.

Also provided are methods of selecting a patient for treatment, whereinthe methods include selecting, identifying, or diagnosing a patienthaving a RET-associated cancer, and selecting the patient for treatmentincluding administration of a therapeutically-effective amount of acompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof. In some embodiments, identifyingor diagnosing a patient as having a RET-associated cancer can include astep of performing an assay on a sample obtained from the patient todetermine whether the patient has a dysregulation of a RET gene, a RETkinase, or expression or activity or level of any of the same, andidentifying or diagnosing a patient determined to have a dysregulationof a RET gene, a RET kinase, or expression or activity or level of anyof the same, as having a RET-associated cancer. In some embodiments, themethod of selecting a patient for treatment can be used as a part of aclinical study that includes administration of various treatments of aRET-associated cancer. In some embodiments, a RET-associated cancer is acancer having one or more RET inhibitor resistance mutations. In someembodiments, the assay is an in vitro assay. For example, an assay thatutilizes the next generation sequencing, immunohistochemistry, or breakapart FISH analysis. In some embodiments, the assay is a regulatoryagency-approved, e.g., FDA-approved, kit. In some embodiments, the assayis a liquid biopsy. In some embodiments, the dysregulation of the RETgene, the RET kinase, or expression or activity or level of any of thesame includes one or more RET inhibitor resistance mutations.

In some embodiments of any of the methods or uses described herein, anassay used to determine whether the patient has a dysregulation of a RETgene, or a RET kinase, or expression or activity or level of any of thesame, using a sample from a patient can include, for example, nextgeneration sequencing, immunohistochemistry, fluorescence microscopy,break apart FISH analysis, Southern blotting, Western blotting, FACSanalysis, Northern blotting, and PCR-based amplification (e.g., RT-PCRand quantitative real-time RT-PCR). As is well-known in the art, theassays are typically performed, e.g., with at least one labelled nucleicacid probe or at least one labelled antibody or antigen-binding fragmentthereof. Assays can utilize other detection methods known in the art fordetecting dysregulation of a RET gene, a RET kinase, or expression oractivity or levels of any of the same (see, e.g., the references citedherein). In some embodiments, the dysregulation of the RET gene, the RETkinase, or expression or activity or level of any of the same includesone or more RET inhibitor resistance mutations. In some embodiments, thesample is a biological sample or a biopsy sample (e.g., aparaffin-embedded biopsy sample) from the patient. In some embodiments,the patient is a patient suspected of having a RET-associated cancer, apatient having one or more symptoms of a RET-associated cancer, and/or apatient that has an increased risk of developing a RET-associatedcancer).

In some embodiments, dysregulation of a RET gene, a RET kinase, or theexpression or activity or level of any of the same can be identifiedusing a liquid biopsy (variously referred to as a fluid biopsy or fluidphase biopsy). See, e.g., Karachialiou et al., “Real-time liquidbiopsies become a reality in cancer treatment”, Ann. Transl. Med.,3(3):36, 2016. Liquid biopsy methods can be used to detect total tumorburden and/or the dysregulation of a RET gene, a RET kinase, or theexpression or activity or level of any of the same. Liquid biopsies canbe performed on biological samples obtained relatively easily from asubject (e.g., via a simple blood draw) and are generally less invasivethan traditional methods used to detect tumor burden and/ordysregulation of a RET gene, a RET kinase, or the expression or activityor level of any of the same. In some embodiments, liquid biopsies can beused to detect the presence of dysregulation of a RET gene, a RETkinase, or the expression or activity or level of any of the same at anearlier stage than traditional methods. In some embodiments, thebiological sample to be used in a liquid biopsy can include, blood,plasma, urine, cerebrospinal fluid, saliva, sputum, broncho-alveolarlavage, bile, lymphatic fluid, cyst fluid, stool, ascites, andcombinations thereof. In some embodiments, a liquid biopsy can be usedto detect circulating tumor cells (CTCs). In some embodiments, a liquidbiopsy can be used to detect cell-free DNA. In some embodiments,cell-free DNA detected using a liquid biopsy is circulating tumor DNA(ctDNA) that is derived from tumor cells. Analysis of ctDNA (e.g., usingsensitive detection techniques such as, without limitation,next-generation sequencing (NGS), traditional PCR, digital PCR, ormicroarray analysis) can be used to identify dysregulation of a RETgene, a RET kinase, or the expression or activity or level of any of thesame.

In some embodiments, ctDNA derived from a single gene can be detectedusing a liquid biopsy. In some embodiments, ctDNA derived from aplurality of genes (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more, or anynumber of genes in between these numbers) can be detected using a liquidbiopsy. In some embodiments, ctDNA derived from a plurality of genes canbe detected using any of a variety of commercially-available testingpanels (e.g., commercially-available testing panels designed to detectdysregulation of a RET gene, a RET kinase, or the expression or activityor level of any of the same). Liquid biopsies can be used to detectdysregulation of a RET gene, a RET kinase, or the expression or activityor level of any of the same including, without limitation, pointmutations or single nucleotide variants (SNVs), copy number variants(CNVs), genetic fusions (e.g., translocations or rearrangements),insertions, deletions, or any combination thereof. In some embodiments,a liquid biopsy can be used to detect a germline mutation. In someembodiments, a liquid biopsy can be used to detect a somatic mutation.In some embodiments, a liquid biopsy can be used to detect a primarygenetic mutation (e.g., a primary mutation or a primary fusion that isassociated with initial development of a disease, e.g., cancer). In someembodiments, a liquid biopsy can be used to detect a genetic mutationthat develops after development of the primary genetic mutation (e.g., aresistance mutation that arises in response to a treatment administeredto a subject). In some embodiments, a dysregulation of a RET gene, a RETkinase, or the expression or activity or level of any of the sameidentified using a liquid biopsy is also present in a cancer cell thatis present in the subject (e.g., in a tumor). In some embodiments, anyof the types of dysregulation of a RET gene, a RET kinase, or theexpression or activity or level of any of the same described herein canbe detected using a liquid biopsy. In some embodiments, a geneticmutation identified via a liquid biopsy can be used to identify thesubject as a candidate for a particular treatment. For example,detection of dysregulation of a RET gene, a RET kinase, or theexpression or activity or level of any of the same in the subject canindicate that the subject will be responsive to a treatment thatincludes administration of a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof.

Liquid biopsies can be performed at multiple times during a course ofdiagnosis, a course of monitoring, and/or a course of treatment todetermine one or more clinically relevant parameters including, withoutlimitation, progression of the disease, efficacy of a treatment, ordevelopment of resistance mutations after administering a treatment tothe subject. For example, a first liquid biopsy can be performed at afirst time point and a second liquid biopsy can be performed at a secondtime point during a course of diagnosis, a course of monitoring, and/ora course of treatment. In some embodiments, the first time point can bea time point prior to diagnosing a subject with a disease (e.g., whenthe subject is healthy), and the second time point can be a time pointafter subject has developed the disease (e.g., the second time point canbe used to diagnose the subject with the disease). In some embodiments,the first time point can be a time point prior to diagnosing a subjectwith a disease (e.g., when the subject is healthy), after which thesubject is monitored, and the second time point can be a time pointafter monitoring the subject. In some embodiments, the first time pointcan be a time point after diagnosing a subject with a disease, afterwhich a treatment is administered to the subject, and the second timepoint can be a time point after the treatment is administered; in suchcases, the second time point can be used to assess the efficacy of thetreatment (e.g., if the genetic mutation(s) detected at the first timepoint are reduced in abundance or are undetectable) or to determine thepresence of a resistance mutation that has arisen as a result of thetreatment. In some embodiments, a treatment to be administered to asubject can include a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof.

In some embodiments, the efficacy of a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,can be determined by assessing the allele frequency of a dysregulationof a RET gene in cfDNA obtained from a patient at different time points,e.g., cfDNA obtained from the patient at a first time point and cfDNAobtained from the patient at a second time point, where at least onedose of a compound of Formula I-IV is administered to the patientbetween the first and second time points. Some embodiments of thesemethods can further include administering to the patient the at leastone dose of the compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof, between the firstand second time points. For example, a reduction (e.g., a 1% to about a99% reduction, a 1% to about a 95% reduction, a 1% to about a 90%reduction, a 1% to about a 85% reduction, a 1% to about a 80% reduction,a 1% to about a 75% reduction, a 1% reduction to about a 70% reduction,a 1% reduction to about a 65% reduction, a 1% reduction to about a 60%reduction, a 1% reduction to about a 55% reduction, a 1% reduction toabout a 50% reduction, a 1% reduction to about a 45% reduction, a 1%reduction to about a 40% reduction, a 1% reduction to about a 35%reduction, a 1% reduction to about a 30% reduction, a 1% reduction toabout a 25% reduction, a 1% reduction to about a 20% reduction, a 1%reduction to about a 15% reduction, a 1% reduction to about a 10%reduction, a 1% to about a 5% reduction, about a 5% to about a 99%reduction, about a 10% to about a 99% reduction, about a 15% to about a99% reduction, about a 20% to about a 99% reduction, about a 25% toabout a 99% reduction, about a 30% to about a 99% reduction, about a 35%to about a 99% reduction, about a 40% to about a 99% reduction, about a45% to about a 99% reduction, about a 50% to about a 99% reduction,about a 55% to about a 99% reduction, about a 60% to about a 99%reduction, about a 65% to about a 99% reduction, about a 70% to about a99% reduction, about a 75% to about a 95% reduction, about a 80% toabout a 99% reduction, about a 90% reduction to about a 99% reduction,about a 95% to about a 99% reduction, about a 5% to about a 10%reduction, about a 5% to about a 25% reduction, about a 10% to about a30% reduction, about a 20% to about a 40% reduction, about a 25% toabout a 50% reduction, about a 35% to about a 55% reduction, about a 40%to about a 60% reduction, about a 50% reduction to about a 75%reduction, about a 60% reduction to about 80% reduction, or about a 65%to about a 85% reduction) in the allele frequency (AF) of thedysregulation of a RET gene in the cfDNA obtained from the patient atthe second time point as compared to the allele frequency (AF) of thedysregulation of a RET gene in the cfDNA obtained from the patient atthe first time point indicates that the compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,was effective in the subject. In some embodiments, the AF is reducedsuch that the level is below the detection limit of the instrument.Alternatively, an increase in the allele frequency (AF) of thedysregulation of a RET gene in the cfDNA obtained from the patient atthe second time point as compared to the allele frequency (AF) of thedysregulation of a RET gene in the cfDNA obtained from the patient atthe first time point indicates that the compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,was not effective in the subject (e.g., the subject has developed aresistance mutation to the compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof).Some embodiments of these methods can further include, administeringadditional doses of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof, to a patient inwhich a compound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, was determined to be effective.Some embodiments of these methods can further include, administering adifferent treatment (e.g., a treatment that does not include theadministration of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, as a monotherapy) to a patient inwhich a compound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, was determined not to beeffective.

In some examples of these methods, the time difference between the firstand second time points can be about 1 day to about 1 year, about 1 dayto about 11 months, about 1 day to about 10 months, about 1 day to about9 months, about 1 day to about 8 months, about 1 day to about 7 months,about 1 day to about 6 months, about 1 day to about 5 months, about 1day to about 4 months, about 1 day to about 3 months, about 1 day toabout 10 weeks, about 1 day to about 2 months, about 1 day to about 6weeks, about 1 day to about 1 month, about 1 day to about 25 days, about1 day to about 20 days, about 1 day to about 15 days, about 1 day toabout 10 days, about 1 day to about 5 days, about 2 days to about 1year, about 5 days to about 1 year, about 10 days to about 1 year, about15 days to about 1 year, about 20 days to about 1 year, about 25 days toabout 1 year, about 1 month to about 1 year, about 6 weeks to about 1year, about 2 months to about 1 year, about 3 months to about 1 year,about 4 months to about 1 year, about 5 months to about 1 year, about 6months to about 1 year, about 7 months to about 1 year, about 8 monthsto about 1 year, about 9 months to about 1 year, about 10 months toabout 1 year, about 11 months to about 1 year, about 1 day to about 7days, about 1 day to about 14 days, about 5 days to about 10 days, about5 day to about 20 days, about 10 days to about 20 days, about 15 days toabout 1 month, about 15 days to about 2 months, about 1 week to about 1month, about 2 weeks to about 1 month, about 1 month to about 3 months,about 3 months to about 6 months, about 4 months to about 6 months,about 5 months to about 8 months, or about 7 months to about 9 months.In some embodiments of these methods, the patient can be previouslyidentified as having a cancer having a dysregulated RET gene (e.g., anyof the examples of a dysregulated RET gene described herein). In someembodiments of these methods, a patient can have been previouslydiagnosed as having any of the types of cancer described herein. In someembodiments of these methods, the patient can have one or moremetastases (e.g., one or more brain metastases).

In some of the above embodiments, the cfDNA comprises ctDNA such asRET-associated ctDNA. For example, the cfDNA is ctDNA such asRET-associated ctDNA. In some embodiments, at least some portion ofcfDNA is determined to be RET-associated ctDNA, for example, a sequencedand/or quantified amount of the total cfDNA is determined to have a RETfusion and/or a RET resistance mutation.

In the field of medical oncology it is normal practice to use acombination of different forms of treatment to treat each patient withcancer. In medical oncology the other component(s) of such conjointtreatment or therapy in addition to compositions provided herein may be,for example, surgery, radiotherapy, and chemotherapeutic agents, such asother kinase inhibitors, signal transduction inhibitors and/ormonoclonal antibodies. For example, a surgery may be open surgery orminimally invasive surgery. Compounds of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereoftherefore may also be useful as adjuvants to cancer treatment, that is,they can be used in combination with one or more additional therapies ortherapeutic agents, for example a chemotherapeutic agent that works bythe same or by a different mechanism of action. In some embodiments, acompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, can be used prior toadministration of an additional therapeutic agent or additional therapy.For example, a patient in need thereof can be administered one or moredoses of a compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof for a period of time and thenunder go at least partial resection of the tumor. In some embodiments,the treatment with one or more doses of a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofreduces the size of the tumor (e.g., the tumor burden) prior to the atleast partial resection of the tumor. In some embodiments, a patient inneed thereof can be administered one or more doses of a compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof for a period of time and under one or more roundsof radiation therapy. In some embodiments, the treatment with one ormore doses of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof reduces the sizeof the tumor (e.g., the tumor burden) prior to the one or more rounds ofradiation therapy.

In some embodiments, a patient has a cancer (e.g., a locally advanced ormetastatic tumor) that is refractory or intolerant to standard therapy(e.g., administration of a chemotherapeutic agent, such as a first RETinhibitor or a multikinase inhibitor, immunotherapy, or radiation (e.g.,radioactive iodine)). In some embodiments, a patient has a cancer (e.g.,a locally advanced or metastatic tumor) that is refractory or intolerantto prior therapy (e.g., administration of a chemotherapeutic agent, suchas a first RET inhibitor or a multikinase inhibitor, immunotherapy, orradiation (e.g., radioactive iodine)). In some embodiments, a patienthas a cancer (e.g., a locally advanced or metastatic tumor) that has nostandard therapy. In some embodiments, a patient is RET-kinase inhibitornaïve. For example, the patient is naïve to treatment with a selectiveRET-kinase inhibitor. In some embodiments, a patient is not RET-kinaseinhibitor naïve.

In some embodiments, a patient has undergone prior therapy. In someembodiments, a patient having NSCLC (e.g, a RET-fusion positive NSCLS)has received treatment with a platinum-based chemotherapy, PD-1/PDL1immunotherapy, or both prior to treatment with a compound of FormulaI-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphform thereof. In some embodiments, a patient having a thyroid cancer(e.g., a RET-fusion positive thyroid cancer) has received treatment withone or more of sorafenib, lenvatinib, and radioactive iodine prior totreatment with a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof. In someembodiments, a patient having a colorectal cancer (e.g., a RET-fusionpositive colorectal cancer) has received treatment with afluoropyrimidine-based chemotherapy, with or without ant-VEGF-directedtherapy or anti-EGFR-directed therapy, prior to treatment with acompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof. In some embodiments, a patienthaving a pancreatic cancer (e.g., a RET-fusion positive pancreaticcancer) has received treatment with one or more of afluoropyrimidine-based chemotherapy, a gemcitabine-based chemotherapy,and a S-1 chemotherapy prior to treatment with a compound of FormulaI-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphform thereof. In some embodiments, a patient having a breast cancer(e.g., a RET-fusion positive breast cancer) has received treatment withone or more of anthracycline, taxane, HER2-directed therapy, andhormonal therapy prior to treatment with a compound of Formula I-IV, ora pharmaceutically acceptable salt, amorphous, or polymorph formthereof. In some embodiments, a patient having a MTC (e.g., a RET-fusionpositive MTC cancer) has received treatment with one or more ofcaboxantinib and vandetanib prior to treatment with a compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof.

In some embodiments of any the methods described herein, the compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof is administered in combination with atherapeutically effective amount of at least one additional therapeuticagent selected from one or more additional therapies or therapeutic(e.g., chemotherapeutic) agents.

Non-limiting examples of additional therapeutic agents include: otherRET-targeted therapeutic agents (i.e. a first or second RET kinaseinhibitor), other kinase inhibitors (e.g., receptor tyrosinekinase-targeted therapeutic agents (e.g., Trk inhibitors or EGFRinhibitors)), signal transduction pathway inhibitors, checkpointinhibitors, modulators of the apoptosis pathway (e.g. obataclax);cytotoxic chemotherapeutics, angiogenesis-targeted therapies,immune-targeted agents, including immunotherapy, and radiotherapy.

In some embodiments, the other RET-targeted therapeutic is a multikinaseinhibitor exhibiting RET inhibition activity. In some embodiments, theother RET-targeted therapeutic inhibitor is selective for a RET kinase.Exemplary RET kinase inhibitors can exhibit inhibition activity (IC₅₀)against a RET kinase of less than about 1000 nM, less than about 500 nM,less than about 200 nM, less than about 100 nM, less than about 50 nM,less than about 25 nM, less than about 10 nM, or less than about 1 nM asmeasured in an assay as described herein. In some embodiments, a RETkinase inhibitors can exhibit inhibition activity (IC₅₀) against a RETkinase of less than about 25 nM, less than about 10 nM, less than about5 nM, or less than about 1 nM as measured in an assay as providedherein.

Non-limiting examples of RET-targeted therapeutic agents (e.g., a firstRET inhibitor or a second RET inhibitor) include alectinib(9-Ethyl-6,6-dimethyl-8-[4-(morpholin-4-yl)piperidin-1-yl]-11-oxo-6,11-dihydro-5H-benzo[b]carbazole-3-carbonitrile);amuvatinib (MP470, HPK56)(N-(1,3-benzodioxol-5-ylmethyl)-4-([1]benzofuro[3,2-d]pyrimidin-4-yl)piperazine-1-carbothioamide);apatinib (YN968D1) (N-[4-(1-cyanocyclopentyl)phenyl-2-(4-picolyl)amino-3-Nicotinamide methanesulphonate);cabozantinib (Cometriq XL-184)(N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)phenyl)-N′-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide);dovitinib (TKI258; GFKI-258; CHIR-258)((3Z)-4-amino-5-fluoro-3-[5-(4-methylpiperazin-1-yl)-1,3-dihydrobenzimidazol-2-ylidene]quinolin-2-one);famitinib(5-[2-(diethylamino)ethyl]-2-[(Z)-(5-fluoro-2-oxo-1H-indol-3-ylidene)methyl]-3-methyl-6,7-dihydro-1H-pyrrolo[3,2-c]pyridin-4-one);fedratinib (SAR302503, TG101348)(N-(2-Methyl-2-propanyl)-3-{[5-methyl-2-({4-[2-(1-pyrrolidinyl)ethoxy]phenyl}amino)-4-pyrimidinyl]amino}benzenesulfonamide);foretinib (XL880, EXEL-2880, GSK1363089, GSK089)(N1′-[3-fluoro-4-[[6-methoxy-7-(3-morpholinopropoxy)-4-quinolyl]oxy]phenyl]-N1-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide);fostamantinib (R788) (2H-Pyrido[3,2-b]-1,4-oxazin-3 (4H)-one,6-[[5-fluoro-2-[(3,4,5-trimethoxyphenyl)amino]-4-pyrimidinyl]amino]-2,2-dimethyl-4-[(phosphonooxy)methyl]—,sodium salt (1:2)); ilorasertib (ABT-348)(1-(4-(4-amino-7-(1-(2-hydroxyethyl)-1H-pyrazol-4-yl)thieno[3,2-c]pyridin-3-yl)phenyl)-3-(3-fluorophenyl)urea);lenvatinib (E7080, Lenvima)(4-[3-chloro-4-cyclopropylaminocarbonyl)aminophenoxy]-7-methoxy-6-quinolinecarboxamide);motesanib (AMG 706)(N-(3,3-Dimethyl-2,3-dihydro-1H-indol-6-yl)-2-[(pyridin-4-ylmethyl)amino]pyridine-3-carboxamide);nintedanib(3-Z-[1-(4-(N-((4-methyl-piperazin-1-yl)-methylcarbonyl)-N-methyl-amino)-anilino)-1-phenyl-methylene]-6-methyoxycarbonyl-2-indolinone);ponatinib (AP24534)(3-(2-Imidazo[1,2-b]pyridazin-3-ylethynyl)-4-methyl-N-[4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl]benzamide);PP242 (torkinib)(2-[4-Amino-1-(1-methylethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl]-1H-indol-5-ol);quizartinib(1-(5-(tert-Butyl)isoxazol-3-yl)-3-(4-(7-(2-morpholinoethoxy)benzo[d]imidazo[2,1-b]thiazol-2-yl)phenyl)urea);regorfenib (BAY 73-4506, stivarga)(4-[4-({[4-Chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)-3-fluorophenoxy]-N-methylpyridine-2-carboxamidehydrate); RXDX-105 (CEP-32496, agerafenib)(1-(3-((6,7-dimethoxyquinazolin-4-yl)oxy)phenyl)-3-(5-(1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol-3-yl)urea);semaxanib (SU5416)((3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)methylidene]-1,3-dihydro-2H-indol-2-one);sitravatinib (MGCD516, MG516)(N-(3-Fluoro-4-{[2-(5-{[(2-methoxyethyl)amino]methyl}-2-pyridinyl)thieno[3,2-b]pyridin-7-yl]oxy}phenyl)-N-(4-fluorophenyl)-1,1-cyclopropanedicarboxamide);sorafenib (BAY 43-9006)(4-[4-[[[[4-chloro-3-(trifluoromethyl)phenyl]amino]carbonyl]amino]phenoxy]-N-methyl-2-pyridinecarboxamide);vandetanib(N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl)methoxy]quinazolin-4-amine);vatalanib (PTK787, PTK/ZK, ZK222584)(N-(4-chlorophenyl)-4-(pyridin-4-ylmethyl)phthalazin-1-amine); AD-57(N-[4-[4-amino-1-(1-methylethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl]phenyl]-N′-[3-(trifluoromethyl)phenyl]-urea);AD-80(1-[4-(4-amino-1-propan-2-ylpyrazolo[3,4-d]pyrimidin-3-yl)phenyl]-3-[2-fluoro-5-(trifluoromethyl)phenyl]urea);AD-81(1-(4-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea);ALW-II-41-27(N-(5-((4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)carbamoyl)-2-methylphenyl)-5-(thiophen-2-yl)nicotinamide);BPR1K871(1-(3-chlorophenyl)-3-(5-(2-((7-(3-(dimethylamino)propoxy)quinazolin-4-yl)amino)ethyl)thiazol-2-yl)urea);CLM3(1-phenethyl-N-(1-phenylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine);EBI-907(N-(2-chloro-3-(1-cyclopropyl-8-methoxy-3H-pyrazolo[3,4-c]isoquinolin-7-yl)-4-fluorophenyl)-3-fluoropropane-1-sulfonamide);NVP-AST-487(N-[4-[(4-ethyl-1-piperazinyl)methyl]-3-(trifluoromethyl)phenyl]-N′-[4-[[6-(methylamino)-4-pyrimidinyl]oxy]phenyl]-urea);NVP-BBT594 (BBT594)(5-((6-acetamidopyrimidin-4-yl)oxy)-N-(4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)indoline-1-carboxamide);PD173955(6-(2,6-dichlorophenyl)-8-methyl-2-(3-methylsulfanylanilino)pyrido[2,3-d]pyrimidin-7-one);PP2(4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine);PZ-1(N-(5-(tert-butyl)isoxazol-3-yl)-2-(4-(5-(1-methyl-1H-pyrazol-4-yl)-1Hbenzo[d]imidazol-1-yl)phenyl)acetamide);RPI-1(1,3-dihydro-5,6-dimethoxy-3-[(4-hydroxyphenyl)methylene]-H-indol-2-one;(3E)-3-[(4-hydroxyphenyl)methylidene]-5,6-dimethoxy-1H-indol-2-one);SGI-7079(3-[2-[[3-fluoro-4-(4-methyl-1-piperazinyl)phenyl]amino]-5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl]-benzeneacetonitrile);SPP86(1-Isopropyl-3-(phenylethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine);SU4984(4-[4-[(E)-(2-oxo-1H-indol-3-ylidene)methyl]phenyl]piperazine-1-carbaldehyde);sunitinb (SU11248)(N-(2-Diethylaminoethyl)-5-[(Z)-(5-fluoro-2-oxo-1H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide);TG101209(N-tert-butyl-3-(5-methyl-2-(4-(4-methylpiperazin-1-yl)phenylamino)pyrimidin-4-ylamino)benzenesulfonamide);Withaferin A((4β,5β,6β,22R)-4,27-Dihydroxy-5,6:22,26-diepoxyergosta-2,24-diene-1,26-dione); XL-999((Z)-5-((1-ethylpiperidin-4-yl)amino)-3-((3-fluorophenyl)(5-methyl-1H-imidazol-2-yl)methylene)indolin-2-one);BPR1J373 (a 5-phenylthiazol-2-ylamine-pyriminide derivative); CG-806(CG'806); DCC-2157; GTX-186; HG-6-63-01((E)-3-(2-(4-chloro-1H-pyrrolo[2,3-b]pyridin-5-yl)vinyl)-N-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)-4-methylbenzamide);SW-01 (Cyclobenzaprine hydrochloride); XMD15-44(N-(4-((4-ethylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)-4-methyl-3-(pyridin-3-ylethynyl)benzamide(generated from structure)); Y078-DM1 (an antibody drug conjugatecomposed of a RET antibody (Y078) linked to a derivative of thecytotoxic agent maytansine); Y078-DM4 (an antibody drug conjugatecomposed of a RET antibody (Y078) linked to a derivative of thecytotoxic agent maytansine); ITRI-305 (D0N5TB, DIB003599); BLU-667 (((1S,4R)—N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methoxy-4-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide);BLU6864; DS-5010; GSK3179106; GSK3352589; NMS-E668; and TAS0286/HM05.

Further examples of RET-targeted therapeutics (e.g., a first RET kinaseinhibitor or a second RET kinase inhibitor) include5-amino-3-(5-cyclopropylisoxazol-3-yl)-1-isopropyl-1H-pyrazole-4-carboxamide;3-(5-cyclopropylisoxazol-3-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-((6,7-Dimethoxyquinazolin-4-yl)amino)-4-fluoro-2-methylphenol;N-(5-(tert-butyl)isoxazol-3-yl)-2-(4-(imidazo[1,2-a]pyridin-6-yl)phenyl)acetamide;N-(5-(tert-butyl)isoxazol-3-yl)-2-(3-(imidazo[1,2-b]pyridazin-6-yloxy)phenyl)acetamide;N-(2-fluoro-5-trifluoromethylphenyl)-N′-{4′-[(2″-benzamido)pyridin-4″-ylamino]phenyl}urea;2-amino-6-{[2-(4-chlorophenyl)-2-oxoethyl]sulfanyl}-4-(3-thienyl)pyridine-3,5-dicarbonitrile;and 3-arylureidobenzylidene-indolin-2-ones.

Additional examples of other RET kinase inhibitors include thosedescribed in U.S. Pat. Nos. 9,150,517 and 9,149,464, and InternationalPublication No. WO 2014075035, all of which are hereby incorporated byreference. For example, in some embodiments the other RET inhibitor is acompound of formula I:

wherein R₁ is C₆-C₂₄alkyl or polyethylene glycol; or a pharmaceuticallyacceptable salt form thereof. In some embodiments, the other RETinhibitor is4-{5-[bis-(chloroethyl)-amino]-1-methyl-1H-benzimidazol-2-yl}butyricacid dodecyl ester.

Additional examples of other RET kinase inhibitors include thosedescribed in International Publication No. WO 2016127074, which ishereby incorporated by reference. For example, in some embodiments, theother RET inhibitor is a compound of Formula (I) or a pharmaceuticallyacceptable salt thereof, wherein:

wherein Rings A and B are each independently selected from aryl,heteroaryl, cycloalkyl and heterocyclyl;

each L¹ and L² is independently selected from a bond, —(C1-C6alkylene)-, —(C2-C6 alkenylene)-, —(C2-C6 alkynylene)-, —(C1-C6haloalkylene)-, —(C1-C6 heteroalkylene)-, —C(O)—, —O—, —S—, —S(O),—S(O)₂—, —N(R¹)—, —O—(C1-C6 alkylene)-, —(C1-C6 alkylene)-O—,—N(R¹)—C(O)—, —C(O)N(R¹)—, —(C1-C6 alkylene)-N(R¹)—, —N(R¹)—(C1-C6alkylene)-, —N(R¹)—C(O)—(C1-C6 alkylene)-, —(C1-C6alkylene)-N(R¹)—C(O)—, —C(O)—N(R¹)—(C1-C6 alkylene)-, —(C1-C6alkylene)-C(O)—N(R¹)—, —N(R¹)—S(O)₂—, —S(O)₂—N(R¹)—, —N(R¹)—S(O)₂—(C1-C6alkylene)-, and —S(O)₂—N(R¹)—(C1-C6 alkylene)-; wherein each alkylene,alkenylene, alkynylene, haloalkylene, and heteroalkylene isindependently substituted with 0-5 occurrences of R′;

each R^(A) and R^(B) is independently selected from C1-C6 alkyl, C1-C6alkoxy, halo, C1-C6 haloalkyl, C1-C6 hydroxyalkyl, C1-C6 heteroalkyl,and —N(R¹)(R¹); wherein each alkyl, alkoxy, haloalkyl, hydroxyalkyl, andhydroxyalkyl is independently substituted with 0-5 occurrences of Ra;

each R^(C) and R^(D) is independently selected from C1-C6 alkyl, C2-C6alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halo, C1-C6 heteroalkyl, C1-C6haloalkyl, C1-C6 haloalkoxy, C1-C6 hydroxyalkyl, cycloalkyl, aryl,heteroaryl, aryloxy, aralkyl, heterocyclyl, heterocyclylalkyl, nitro,cyano, —C(O)R¹, —OC(O)R¹, —C(O)OR¹, —(C1-C6 alkylene)-C(O)R¹, —SR¹,—S(O)₂R¹, —S(O)₂—N(R¹)(R¹), —(C1-C6 alkylene)-S(O)₂R¹, —(C1-C6alkylene)-S(O)₂—N(R¹)(R¹), —N(R¹)(R¹)—C(O)—N(R¹)(R¹)—N(R¹)—C(O)R¹,—N(R¹)—C(O)OR¹, —(C1-C6 alkylene)-N(R¹)—C(O)R¹, —N(R¹)S(O)₂R¹, and—P(O)(R¹)(R¹); wherein each of alkyl, alkenyl, alkynyl, alkoxy,heteroalkyl, haloalkyl, haloalkoxy, hydroxyalkyl, cycloalkyl, aryl,heteroaryl, aryloxy, aralkyl, heterocyclyl, and heterocyclylalkyl isindependently substituted with 0-5 occurrences of R^(a); or 2 R^(C) or 2R^(D) together with the carbon atom(s) to which they are attached form acycloalkyl or heterocyclyl ring independently substituted with 0-5occurrences of R^(a);

each R¹ is independently selected from hydrogen, hydroxyl, halo, thiol,C1-C6 alkyl, C1-C6 thioalkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6hydroxyalkyl, C1-C6 heteroalkyl, cycloalkyl, cycloalkylalkyl,heteroarylalkyl, heterocyclyl, and heterocyclylalkyl, wherein each ofalkyl, thioalkyl, alkoxy, haloalkyl, hydroxyalkyl, heteroalkyl,cycloalkyl, cycloalkylalkyl, heteroarylalkyl, heterocyclyl, andheterocyclylalkyl is independently substituted with 0-5 occurrences ofR^(b), or 2 R¹ together with the atom(s) to which they are attached forma cycloalkyl or heterocyclyl ring independently substituted with 0-5occurrences of R^(b);

each R^(a) and R^(b) is independently C1-C6 alkyl, halo, hydroxyl, C1-C6haloalkyl, C1-C6 heteroalkyl, C1-C6 hydroxyalkyl, C1-C6 alkoxy,cycloalkyl, heterocyclyl, or cyano, wherein each of alkyl, haloalkyl,heteroalkyl, hydroxyalkyl, alkoxy, cycloalkyl and heterocyclyl isindependently substituted with 0-5 occurrences of R′;

each R′ is C1-C6 alkyl, C1-C6 heteroalkyl, halo, hydroxyl, C1-C6haloalkyl, C1-C6 hydroxyalkyl, cycloalkyl or cyano; or 2 R′, togetherwith the atom(s) to which they are attached form a cycloalkyl orheterocyclyl ring;

m is 0, 1, 2, or 3;

n is 0, 1, or 2; and

p and q are each independently 0, 1, 2, 3, or 4. For example, a RETinhibitor can be selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

Additional examples of other RET kinase inhibitors include thosedescribed in International Publication No. WO 2016075224, which ishereby incorporated by reference. For example, in some embodiments, theother RET inhibitor is a compound of Formula (II) or a pharmaceuticallyacceptable salt thereof, wherein:

R1 and R2 are independently hydrogen or an optionally substituted groupselected from straight or branched (C₁-C₆) alkyl, (C₃-C₆) cycloalkyl andCOR′, wherein R′ is an optionally substituted group selected fromstraight or branched (C₁-C₆) alkyl and (C₃-C₆) cycloalkyl;

R3 is hydrogen or an optionally substituted group selected from straightor branched (C₁-C₆) alkyl, (C₂-C₆) alkenyl, (C₂-C₆) alkynyl, (C₃-C₆)cycloalkyl, aryl, heteroaryl and a 3- to 7-membered heterocyclyl ring;

R4 is hydrogen or an optionally substituted group selected from straightor branched (C₁-C₆) alkyl, (C₂-C₆) alkenyl, aryl, heteroaryl orheterocyclyl;

A is a 5- or 6-membered heteroaryl ring or a phenyl ring;

B is a 5- or 6-membered ring selected from heteroaryl, (C₅-C₆)cycloalkyl and heterocyclyl ring or a phenyl ring; wherein ring A andring B are fused together to form a bicyclic system comprising a6-membered aromatic or 5- to 6-membered heteroaromatic ring fused with a6-membered aromatic or 5- to 6-membered heteroaromatic, (C₅-C₆)cycloalkyl or heterocyclyl ring;

Y is carbon or nitrogen;

X is hydrogen, halogen, hydroxyl, cyano or an optionally substitutedgroup selected from straight or branched (C₁-C₆) alkyl and (C₁-C₆)alkoxyl; and

R5 and R6 are independently hydrogen or an optionally substituted groupselected from straight or branched (C₁-C₆) alkyl, (C₃-C₆) cycloalkyl,heterocyclyl, aryl and heteroaryl.

Additional examples of other RET kinase inhibitors include thosedescribed in International Publication No. WO 2015079251, which ishereby incorporated by reference. For example, in some embodiments, theother RET inhibitor is a compound of Formula (III) or a pharmaceuticallyacceptable salt or solvate thereof, wherein:

X is NH, NR_(x), 0 or S, wherein R_(x) is (1-3C)alkyl;

R₁ is selected from halo (e.g., fluoro, chloro, or bromo),trifluoromethyl, (1-4C)alkyl (e.g., methyl), (1-4C)alkoxy or(3-6C)cycloalkyl, wherein an alkyl, alkoxy or cycloalkyl group isoptionally substituted with one or more fluoro;

R₂ is selected from hydrogen, halo (e.g., fluoro, chloro or bromo),hydroxyl, cyano, trifluoromethyl, trifluoromethoxy, (1-6C)alkyl (e.g.,methyl), (3-8C)cycloalkyl, or (1-4C)alkoxy (e.g., OMe), wherein analkyl, cycloalkyl or alkoxy group is optionally substituted with one ormore fluoro;

R₃ is selected from hydrogen, halo (e.g. fluoro, chloro or bromo),hydroxyl, cyano, trifluoromethyl, trifluoromethoxy, (1-6C)alkyl (e.g.,methyl), (3-8C)cycloalkyl, or (1-4C)alkoxy (e.g., OMe), wherein analkyl, cycloalkyl or alkoxy group is optionally substituted with one ormore fluoro;

R₄ is selected from hydrogen, halo (e.g., fluoro, chloro or bromo),hydroxyl, cyano, trifluoromethyl, trifluoromethoxy, (1-6C)alkyl (e.g.,methyl), (3-8C)cycloalkyl, or (1-4C)alkoxy (e.g., OMe), wherein analkyl, cycloalkyl or alkoxy group is optionally substituted with one ormore fluoro;

R₅ is selected from hydrogen or a group defined by the formula—O-L₅-X₅-Q₅;

wherein

-   -   L₅ is absent or a linear or branched (1-4C)alkylene;    -   X₅ is absent or —C(O)O—, —O—, —C(O)—, —OC(O)—, —CH(QR_(5L))—,        —N(R^(j))—, —N(R_(5L))—C(O)—, —N(R_(5L))—C(O)O—,        —C(O)—N(R_(5L))—, —S—, —SO—, —SO₂—, —S(O)₂N(R_(5L))—, or        —N(R_(5L))SO₂— wherein R_(5L) is selected from hydrogen or        methyl; and    -   Q₅ is (1-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl,        (3-8C)cycloalkyl, (3-8C)cycloalkyl-(1-4C)alkyl, aryl,        aryl-(1-4C)alkyl, heteroaryl, heteroaryl-(1-4C)alkyl,        heterocyclyl or heterocyclyl-(1-4C)alkyl;

R₆ is selected from hydrogen, or a group defined by the formula:—O-L₆-X₆-Q₆

wherein

-   -   L₆ is absent or a linear or branched (1-4C)alkylene;    -   X₆ is absent or selected from —O—, —C(O)—, —C(O)O—, —OC(O)—,        —CH(OR_(6L))—, —N(R_(6L)), —N(R_(6L))—C(O)—, —N(R_(6L))—C(O)O—,        —C(O)—N(R_(6L))—, —S—, —SO—, —SO₂—, —S(O)₂N(R_(6L))—, or        —N(R_(6L))SO₂— wherein R_(6L) is selected from hydrogen or        (1-3C)alkyl;    -   Q₆ is hydrogen, (1-8C)alkyl, (2-8C)alkenyl, (2-8C)alkynyl,        (3-8C)cycloalkyl, (3-8C)cycloalkyl-(1-6C)alkyl, aryl,        aryl-(1-6C)alkyl, heteroaryl, heteroaryl-(1-6C)alkyl,        heterocyclyl, heterocyclyl-(1-6C)alkyl,    -   or Q₆ and R_(L6) are linked such that, together with the        nitrogen atom to which they are attached, they form a        heterocyclic ring;    -   wherein R₆ is optionally substituted (e.g. substituted on L₆        and/or Q₆) with one or more (1-6C)alkyl, (1-6C)alkanoyl,        OR_(6X), SR_(6X), S(O)R_(6X), S(O)₂R_(6X), C(O)OR_(6X) or        C(O)NR_(6X)R′_(6X), wherein R_(6X) and R′_(6X) are independently        hydrogen, (1-8C)alkyl, or R_(6X) and R′_(6X) are linked such        that, together with the nitrogen atom to which they are        attached, they form a heterocyclic ring; and

R₇ is selected from hydrogen, (1-6C)alkoxy, or a group defined by theformula:—O-L₇-X₇-Q₇-

wherein

-   -   L₇ is absent or a linear or branched (1-4C)alkylene;    -   X₇ is absent or selected from —O—, —C(O)—, —C(O)O—, —OC(O)—,        —CH(OR_(6L))—, —N(R_(7L))—, —N(R_(7L))—C(O)—, —N(R_(7L))—C(O)O—,        —C(O)—N(R_(7L))—, —S—, —SO—, —SO₂—, —S(O)₂N(R_(7L))—, or        —N(R_(7L))SO₂— wherein R_(7L) is selected from hydrogen or        (1-3C)alkyl;    -   Q₇ is hydrogen, (1-8C)alkyl, (2-8C)alkenyl, (2-8C)alkynyl,        (3-8C)cycloalkyl, (3-8C)cycloalkyl-(1-6C)alkyl, aryl,        aryl-(1-6C)alkyl, heteroaryl, heteroaryl-(1-6C)alkyl,        heterocyclyl, heterocyclyl-(1-6C)alkyl,    -   or Q₇ and R_(7L) are linked such that, together with the        nitrogen atom to which they are attached, they form a        heterocyclic ring;    -   wherein R₇ is optionally substituted (e.g., substituted on L₇        and/or Q₇) with one or more halo, hydroxyl, nitro, cyano,        (1-8C)alkyl, (1-8C)alkanoyl, OR_(7X), SR_(7X), S(O)R_(7X),        S(O)₂R_(7X), C(O)OR_(7X) or C(O)NR_(7X)R′_(7X), wherein R_(7X)        and R′_(7X) are independently hydrogen, (1-8C)alkyl, or R_(7X)        and R′_(7X) are linked such that, together with the nitrogen        atom to which they are attached, they form a heterocyclic ring;        or    -   R7 is optionally substituted with one or more groups selected        from oxo, (1-4C)haloalkyl, (1-4C)hydroxyalkyl, C(O)R_(7y) or        NR_(7y)R′_(7y), wherein R_(7y) and R′_(7y) are independently        hydrogen or (1-8C)alkyl.

Additional examples of other RET kinase inhibitors include thosedescribed in International Publication No. WO2017178845, which is herebyincorporated by reference. For example, in some embodiments, the otherRET inhibitor is a compound of Formula (IV) or a pharmaceuticallyacceptable salt thereof, wherein:

HET is selected from one of the following:

wherein

denotes the point of attachment;R₁ is selected from hydrogen, (1-4C)haloalkyl, (1-4C)haloalkoxy or agroup of the formula:-L-Y-Qwherein:

L is absent or (1-5C)alkylene optionally substituted by one or moresubstituents selected from (1-2C)alkyl or oxo;

Y is absent or O, S, SO, SO₂, N(R_(a)), C(O), C(O)O, OC(O),C(O)N(R_(a)), N(R_(a))C(O), N(R_(a))C(O)N(R_(b)), N(R_(a))C(O)O,OC(O)N(R_(a)), S(O)₂N(R_(a)), or N(R_(a))SO₂, wherein R_(a) and R_(b)are each independently selected from hydrogen or (1-4C)alkyl; and

Q is hydrogen, (1-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl,(3-10C)cycloalkyl, (3-10C)cycloalkenyl, heteroaryl or heterocyclyl;wherein Q is optionally further substituted by one or more substituentgroups independently selected from (1-4C)alkyl, halo, (1-4C)haloalkyl,(1-4C)haloalkoxy, amino, (1-4C)aminoalkyl, cyano, hydroxy, carboxy,carbamoyl, sulphamoyl, mercapto, ureido, NR_(c)R_(d), OR_(c), C(O)R_(c),C(O)OR_(c), OC(O)R_(c), C(O)N(R_(d))R_(c), N(R_(d))C(O)R_(c),S(O)_(p)R_(c) (where p is 0, 1 or 2), SO₂N(R_(d))R_(c),N(R_(d))SO₂R_(c), Si(R_(e))(R_(d))R_(c) or (CH₂)_(q)NR_(c)R_(d) (where qis 1, 2 or 3); wherein R_(c), R_(d) and R_(e) are each independentlyselected from hydrogen, (1-6C)alkyl or (3-6C)cycloalkyl; or R_(c) andR_(d) are linked such that, together with the nitrogen atom to whichthey are attached, they form a 4-7 membered heterocyclic ring which isoptionally substituted by one or more substituents selected from(1-4C)alkyl, halo, (1-4C)haloalkyl, (1-4C)haloalkoxy, (1-4C)alkoxy,(1-4C)alkylamino, amino, cyano or hydroxy; or Q is optionallysubstituted by a group of the formula:-L₁-L_(Q1)-W₁

wherein:

-   -   L₁ is absent or (1-3C)alkylene optionally substituted by one or        more substituents selected from (1-2C)alkyl or oxo;    -   L_(Q1) is absent or selected from O, S, SO, SO₂, N(R_(f)), C(O),        C(O)O, OC(O), C(O)N(R_(f)), N(R_(f))C(O), N(R_(f))C(O)N(R_(g)),        N(R_(f))C(O)O, OC(O)N(R_(f)), S(O)₂N(R_(f)), or N(R_(f))SO₂,        wherein R_(f) and R_(g) are each independently selected from        hydrogen or (1-2C)alkyl; and    -   W₁ is hydrogen, (1-6C)alkyl, aryl, aryl(1-2C)alkyl,        (3-8C)cycloalkyl, (3-8C)cycloalkenyl, heteroaryl or        heterocyclyl; wherein W₁ is optionally substituted by one or        more substituents selected from (1-4C)alkyl, halo,        (1-4C)haloalkyl, (1-4C)haloalkoxy, (1-4C)alkoxy,        (1-4C)alkylamino, amino, cyano, hydroxy, carboxy, carbamoyl,        sulphamoyl, mercapto, ureido, aryl, heteroaryl, heterocycyl,        (3-6C)cycloalkyl, NR_(h)R_(i), OR_(h), C(O)R_(h), C(O)OR_(h),        OC(O)R_(h), C(O)N(R_(i))R_(h), N(R_(i))C(O)R_(h), S(O)_(r)R_(h)        (where r is 0, 1 or 2), SO₂N(R_(i))R_(h), N(R_(i))SO₂R_(h) or        (CH₂)_(s)NR_(i)R_(h) (where s is 1, 2 or 3); wherein R_(h) and        R_(i) are each independently selected from hydrogen, (1-4C)alkyl        or (3-6C)cycloalkyl;

R_(1a) and R_(1b) are each selected from H, (1-4C)alkyl, halo,(1-4C)haloalkyl, (1-4C)haloalkoxy, (1-4C)alkoxy, (1-4C)alkylamino,amino, cyano, hydroxy, carboxy, carbamoyl, sulphamoyl or mercapto;

W is selected from 0, S or NR_(W1), wherein R_(W1) is selected from H or(1-2C)alkyl;

X₁, X₂, X₃ and X₄ are independently selected from CH, CR₂ or N;

R₂ is selected from hydrogen, halo, (1-4C)alkyl, (1-4C)alkoxy,(1-4C)haloalkyl, (1-4C)haloalkoxy, amino, cyano, nitro, aryl,heteroaryl, heterocyclyl, cycloalkyl, (2-4C)alkynyl, NR_(j)R_(k),OR_(j), C(O)R_(j), C(O)OR_(j), OC(O)R_(j), C(O)N(R_(k))R_(j),N(R_(k))C(O)R_(j), N(R_(k))C(O)N(R_(j)), S(O)_(r1)R_(k) (where r₁ is 0,1 or 2), SO₂N(R_(j))R_(k), N(R_(j))SO₂R_(k) or (CH₂)_(v)NR_(j)R_(k)(where v is 1, 2 or 3); wherein and R_(k) are each independentlyselected from hydrogen or (1-4C)alkyl; and wherein said (1-4C)alkyl,aryl, heteroaryl, heterocycyl or cycloalkyl is optionally substituted byone or more substituents selected from halo, (1-4C)alkyl, (1-4C)alkoxy,(1-4C)haloalkyl, (1-4C)haloalkoxy, amino, cyano, nitro, phenyl,(2-4C)alkynyl, NR_(j1)R_(k1), OR_(j1), C(O)R_(j1), C(O)OR_(j1),OC(O)R_(j1), C(O)N(R_(k1))R_(j1), N(R_(k1))C(O)R_(j1), S(O)_(r2)R_(h)(where r₂ is 0, 1 or 2), SO₂N(R_(j1))R_(k1), N(R_(j1))SO₂R_(k1) or(CH₂)_(v1)NR_(j1)R_(k1) (where v₁ is 1, 2 or 3); and wherein R_(j1) andR_(k1) are each independently selected from hydrogen or (1-4C)alkyl; and

R₃ is selected from halo, (1-4C)alkyl, (1-4C)alkoxy, (1-4C)haloalkyl,(1-4C)haloalkoxy, amino, cyano, nitro, (2-4C)alkynyl, NR_(l)R_(m),OR_(l), C(O)R_(l), C(O)OR_(l), OC(O)R_(l), C(O)N(R_(m))R_(l),N(R_(m))C(O)R_(l), or (CH₂)_(y)NR_(l)R_(m) (where y is 1, 2 or 3);wherein said (1-4C)alkyl is optionally substituted by one or moresubstituents selected from amino, hydroxy, (1-2C)alkoxy or halo; andwherein R_(l) and R_(m) are each independently selected from hydrogen or(1-4C)alkyl.

Additional examples of other RET kinase inhibitors include thosedescribed in International Publication No. WO2017178844, which is herebyincorporated by reference. For example, in some embodiments, the otherRET inhibitor is a compound of Formula (V) or a pharmaceuticallyacceptable salt thereof, wherein:

HET is selected from one of the following:

wherein

denotes the point of attachment;R₁ is selected from hydrogen, (1-4C)haloalkyl, (1-4C)haloalkoxy or agroup of the formula:-L-Y-Qwherein:

L is absent or (1-5C)alkylene optionally substituted by one or moresubstituents selected from (1-2C)alkyl or oxo;

Y is absent or O, S, SO, SO₂, N(R_(a)), C(O), C(O)O, OC(O),C(O)N(R_(a)), N(R_(a))C(O), N(R_(a))C(O)N(R_(b)), N(R_(a))C(O)O,OC(O)N(R_(a)), S(O)₂N(R_(a)), or N(R_(a))SO₂, wherein R_(a) and R_(b)are each independently selected from hydrogen or (1-4C)alkyl; and

Q is hydrogen, (1-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl,(3-10C)cycloalkyl, (3-10C)cycloalkenyl, heteroaryl or heterocyclyl;wherein Q is optionally further substituted by one or more substituentgroups independently selected from (1-4C)alkyl, halo, (1-4C)haloalkyl,(1-4C)haloalkoxy, amino, (1-4C)aminoalkyl, cyano, hydroxy, carboxy,carbamoyl, sulphamoyl, mercapto, ureido, NR_(c)R_(d), OR_(c), C(O)R_(c),C(O)OR_(c), OC(O)R_(c), C(O)N(R_(d))R_(c), N(R_(d))C(O)R_(c),S(O)_(y)R_(c) (where y is 0, 1 or 2), SO₂N(R_(d))R_(c),N(R_(d))SO₂R_(c), Si(R_(d))(R_(c))R_(e) or (CH₂)_(z)NR_(c)R_(d) (where zis 1, 2 or 3); wherein R_(e), R_(d) and R_(e) are each independentlyselected from hydrogen, (1-6C)alkyl or (3-6C)cycloalkyl; or R_(e) andR_(d) can be linked such that, together with the nitrogen atom to whichthey are attached, they form a 4-7 membered heterocyclic ring which isoptionally substituted by one or more substituents selected from(1-4C)alkyl, halo, (1-4C)haloalkyl, (1-4C)haloalkoxy, (1-4C)alkoxy,(1-4C)alkylamino, amino, cyano or hydroxyl; or

Q is optionally substituted by a group of the formula:-L₁-L_(Q1)-Z₁

-   -   wherein:    -   L₁ is absent or (1-3C)alkylene optionally substituted by one or        more substituents selected from (1-2C)alkyl or oxo;    -   L_(Q1) is absent or selected from O, S, SO, SO₂, N(R_(f)), C(O),        C(O)O, OC(O), C(O)N(R_(f)), N(R_(f))C(O), N(R_(g))C(O)N(R_(f)),        N(R_(f))C(O)O, OC(O)N(R_(f)), S(O)₂N(R_(f)), or N(R_(f))SO₂,        wherein R_(f) and R_(g) are each independently selected from        hydrogen or (1-2C)alkyl; and    -   Z₁ is hydrogen, (1-6C)alkyl, aryl, aryl(1-2C)alkyl,        (3-8C)cycloalkyl, (3-8C)cycloalkenyl, heteroaryl or        heterocyclyl; wherein Z₁ is optionally substituted by one or        more substituents selected from (1-4C)alkyl, halo,        (1-4C)haloalkyl, (1-4C)haloalkoxy, (1-4C)alkoxy,        (1-4C)alkylamino, amino, cyano, hydroxy, carboxy, carbamoyl,        sulphamoyl, mercapto, ureido, aryl, heteroaryl, heterocycyl,        (3-6C)cycloalkyl, NR_(h)R_(i), OR_(h), C(O)R_(h), C(O)OR_(h),        OC(O)R_(h), C(O)N(R_(i))R_(h), N(R_(i))C(O)R_(h), S(O)_(ya)R_(h)        (where y^(a) is 0, 1 or 2), SO₂N(R_(i))R_(h), N(R_(i))SO₂R_(h)        or (CH₂)_(za)NR_(i)R_(h) (where z^(a) is 1, 2 or 3); wherein        R_(h) and R_(i) are each independently    -   selected from hydrogen, (1-4C)alkyl or (3-6C)cycloalkyl;

R_(1a) and R_(1b) are each selected from hydrogen, (1-4C)alkyl, halo,(1-4C)haloalkyl, (1-4C)haloalkoxy, (1-4C)alkoxy, (1-4C)alkylamino,amino, cyano, hydroxy, carboxy, carbamoyl, sulphamoyl or mercapto;

W is selected from O, S or NR_(j), wherein R_(j) is selected from H or(1-2C)alkyl;

X₁ and X₂ are each independently selected from N or CR_(k);

wherein

-   -   R_(k) is selected from hydrogen, halo, (1-4C)alkyl,        (1-4C)alkoxy, amino, (1-4C)alkylamino, (1-4C)dialkylamino,        cyano, (2C)alkynyl, C(O)R_(k1), C(O)OR_(k1), OC(O)R_(k1),        C(O)N(R_(k2))R_(k1), N(R_(k2))C(O)R_(k1), S(O)_(yb)R_(k1) (where        y^(b) is 0, 1 or 2), SO₂N(R_(k2))R_(k1), N(R_(k2))SO₂R_(k1) or        (CH₂)_(zb)NR_(k1)R_(k2) (where z^(b) is 1, 2 or 3); wherein said        (1-4C)alkyl is optionally substituted by one or more        substituents selected from amino, hydroxy, (1-2C)alkoxy or halo;        and    -   R_(k1) and R_(k2) are each independently selected from hydrogen        or (1-4C)alkyl;

X₃ is selected from N or CR_(m);

wherein

-   -   R_(m) is selected from hydrogen, halo, (1-4C)alkyl,        (1-4C)alkoxy, amino, (1-4C)alkylamino, (1-4C)dialkylamino,        cyano, (2C)alkynyl, C(O)R_(m1), C(O)OR_(m1), OC(O)R_(m1),        C(O)N(R_(m2))R_(m1), N(R_(m2))C(O)R_(m1), S(O)_(yc)R_(m1) (where        y^(c) is 0, 1 or 2), SO₂N(R_(m2))R_(m1), N(R_(m2))SO₂R_(m)1 or        (CH₂)_(zc)NR_(m1)R_(m2) (where zc is 1, 2 or 3); wherein said        (1-4C)alkyl is optionally substituted by one or more        substituents selected from amino, hydroxy, (1-2C)alkoxy or halo;        and    -   R_(m1) and R_(m1) are each independently selected from hydrogen        or (1-4C)alkyl;

R_(o) is selected from halo, (1-4C)alkyl, (1-4C)alkoxy, amino,(1-4C)alkylamino, (1-4C)dialkylamino, cyano, (2C)alkynyl, C(O)R_(o1),C(O)OR_(o1), OC(O)R_(o1), C(O)N(R_(o2))R_(o1), N(R_(o2))C(O)R_(o1),S(O)_(yd)R_(o1) (where y^(d) is 0, 1 or 2), SO₂N(R_(o2))R_(o1),N(R_(o2))SO₂R_(o1) or (CH₂)_(zd)NR_(o1)R_(o2) (where Z^(d) is 1, 2 or3); wherein said (1-4C)alkyl is optionally substituted by one or moresubstituents selected from amino, hydroxy, (1-2C)alkoxy or halo; and

R_(o1) and R_(o2) are each independently selected from hydrogen or(1-4C)alkyl;

R₂ is selected from hydrogen, (1-4C)alkyl or a group of the formula:-L₂-Y₂-Q₂

wherein:

-   -   L₂ is absent or (1-3C)alkylene optionally substituted by one or        more substituents selected from (1-2C)alkyl or oxo;    -   Y₂ is absent or C(O), C(O)O, C(O)N(R_(p)), wherein R_(p) is        selected from hydrogen or (1-4C)alkyl; and    -   Q₂ is hydrogen, (1-6C)alkyl, aryl, (3-8C)cycloalkyl,        (3-8C)cycloalkenyl, heteroaryl or heterocyclyl; wherein Q₂ is        optionally further substituted by one or more substituent groups        independently selected from (1-4C)alkyl, halo, (1-4C)haloalkyl,        (1-4C)haloalkoxy, amino, cyano, hydroxy, carboxy, carbamoyl,        sulphamoyl, NR_(q)R_(r), OR_(q), wherein R_(q) and R_(r) are        each independently selected from hydrogen, (1-4C)alkyl or        (3-6C)cycloalkyl; R₃ is selected from a group of the formula:        —Y₃-Q₃

wherein:

-   -   Y₃ is C(O), C(O)N(R_(y)), C(O)N(R_(y))O, N(R_(y))(O)C, C(O)O,        OC(O), N(R_(y))C(O)N(R_(y1)), SO₂N(R_(y)), N(R_(y))SO₂,        oxazolyl, triazolyl, oxadiazolyl, thiazolyl, imidazolyl,        thiadiazolyl, pyridinyl, pyrazolyl, pyrrolyl or tetrazolyl,        wherein R_(y) and R_(y1) are independently selected from        hydrogen or (1-2C)alkyl; and    -   Q₃ is hydrogen, (1-6C)alkyl, aryl, aryl(1-2C)alkyl,        (3-8C)cycloalkyl, (3-8C)cycloalkenyl, heteroaryl or        heterocyclyl; wherein Q₃ is optionally further substituted by        one or more substituent groups independently selected from        (1-4C)alkyl, halo, (1-4C)haloalkyl, (1-4C)haloalkoxy, amino,        cyano, hydroxy, carboxy, carbamoyl, sulphamoyl, NR_(z)R_(aa),        OR_(z), wherein R_(z) and R_(aa) are each independently selected        from hydrogen, (1-4C)alkyl or (3-6C)cycloalkyl; or Q₃ is        optionally substituted by a group of the formula:        -L₄-L_(Q4)-Z₄

wherein:

-   -   L₄ is absent or (1-3C)alkylene optionally substituted by one or        more substituents selected from (1-2C)alkyl or oxo;    -   L_(Q4) is absent or selected from or O, S, SO, SO₂, N(R_(ab)),        C(O), C(O)O, OC(O), C(O)N(R_(ab)), N(R_(ab))C(O),        N(R_(ac))C(O)N(R_(ab)), N(R_(ab))C(O)O, OC(O)N(R_(ab)),        S(O)₂N(R_(ab)), or N(R_(ab))SO₂, wherein R_(ab) and R_(ac) are        each independently selected from hydrogen or (1-2C)alkyl; and    -   Z₄ is hydrogen, (1-6C)alkyl, aryl, aryl(1-2C)alkyl,        (3-8C)cycloalkyl, (3-8C)cycloalkenyl, heteroaryl or        heterocyclyl; wherein Z₄ is optionally substituted by one or        more substituents selected from (1-4C)alkyl, halo,        (1-4C)haloalkyl, (1-4C)haloalkoxy, (1-4C)alkoxy,        (1-4C)alkylamino, amino, cyano, hydroxy, carboxy, carbamoyl,        sulphamoyl, mercapto, ureido, aryl, heteroaryl, heterocycyl,        (3-6C)cycloalkyl, NR_(ad)R_(ae), OR_(ad), C(O)R_(ad),        C(O)OR_(ad), OC(O)R_(ad), C(O)N(R_(ae))R_(ad),        N(R_(ae))C(O)R_(ad), S(O)_(ye)R_(ad) (where y^(e) is 0, 1 or 2),        SO₂N(R_(ae))R_(ad), N(R_(ae))SO₂R_(ad) or        (CH₂)_(ze)NR_(ad)R_(ae) (where z^(e) is 1, 2 or 3); wherein        R_(ad) and R_(ae) are each independently selected from hydrogen,        (1-4C)alkyl or (3-6C)cycloalkyl; or    -   Q₃ and R_(y) are linked such that, together with the nitrogen        atom to which they are attached, they form a 4-7 membered        heterocyclic ring which is optionally substituted by one or more        substituents selected from (1-4C)alkyl, halo, (1-4C)haloalkyl,        (1-4C)haloalkoxy, (1-4C)alkoxy, (1-4C)alkylamino, amino, cyano        or hydroxyl;

with the proviso that only one or two of X₁, X₂ or X₃ can be N.

Additional examples of other RET kinase inhibitors include thosedescribed in International Publication No. WO 2017145050, which ishereby incorporated by reference. For example, in some embodiments, theother RET has the Formula (VI) or is a pharmaceutically acceptable saltthereof.

Additional examples of other RET kinase inhibitors include thosedescribed in International Publication No. WO 2016038552 is herebyincorporated by reference. For example, in some embodiments, the otherRET has the Formula (VII), or the Formula (VIII), or is apharmaceutically acceptable salt thereof.

Yet other therapeutic agents include RET inhibitors such as thosedescribed, for example, in U.S. Pat. Nos. 10,030,005; 9,738,660;9,801,880; 9,682,083; 9,789,100; 9,550,772; 9,493,455; 9,758,508;9,604,980; 9,321,772; 9,522,910; 9,669,028; 9,186,318; 8,933,230;9,505,784; 8,754,209; 8,895,744; 8,629,135; 8,815,906; 8,354,526;8,741,849; 8,461,161; 8,524,709; 8,129,374; 8,686,005; 9,006,256;8,399,442; 7,795,273; 7,863,288; 7,465,726; 8,552,002; 8,067,434;8,198,298; 8,106,069; 6,861,509; 8,299,057; 9,150,517; 9,149,464;8,299,057; and 7,863,288; U.S. Publication Nos. 2018/0009818;2018/0009817; 2017/0283404; 2017/0267661; 2017/0298074; 2017/0114032;2016/0009709; 2015/0272958; 2015/0238477; 2015/0099721; 2014/0371219;2014/0137274; 2013/0079343; 2012/0283261; 2012/0225057; 2012/0065233;2013/0053370; 2012/0302567; 2011/0189167; 2016/0046636; 2013/0012703;2011/0281841; 2011/0269739; 2012/0271048; 2012/0277424; 2011/0053934;2011/0046370; 2010/0280012; 2012/0070410; 2010/0081675; 2010/0075916;2011/0212053; 2009/0227556; 2009/0209496; 2009/0099167; 2010/0209488;2009/0012045; 2013/0303518; 2008/0234267; 2008/0199426; 2010/0069395;2009/0312321; 2010/0173954; 2011/0195072; 2010/0004239; 2007/0149523;2017/0281632; 2017/0226100; 2017/0121312; 2017/0096425; 2017/0044106;2015/0065468; 2009/0069360; 2008/0275054; 2007/0117800; 2008/0234284;2008/0234276; 2009/0048249; 2010/0048540; 2008/0319005; 2009/0215761;2008/0287427; 2006/0183900; 2005/0222171; 2005/0209195; 2008/0262021;2008/0312192; 2009/0143399; 2009/0130229; 2007/0265274; 2004/0185547;and 2016/0176865; and International Publication Nos. WO 2018/149382; WO2018/136796; WO 2017/079140; WO 2017/145050; WO 2017/097697; WO2017/049462; WO 2017/043550; WO 2017/027883; WO 2017/013160; WO2017/009644; WO 2016/168992; WO 2016/137060; WO 2016/127074; WO2016/075224; WO 2016/038552; WO 2015/079251; WO 2014/086284; WO2013/042137; WO 2013/036232; WO 2013/016720; WO 2012/053606; WO2012/047017; WO 2007/109045; WO 2009/042646; WO 2009/023978; WO2009/017838; WO 2017/178845; WO 2017/178844; WO 2017/146116; WO2017/026718; WO 2016/096709; WO 2007/057397; WO 2007/057399; WO2007/054357; WO 2006/130613; WO 2006/089298; WO 2005/070431; WO2003/020698; WO 2001/062273; WO 2001/016169; WO 1997/044356; WO2007/087245; WO 2005/044835; WO 2014/075035; and WO 2016/038519; and J.Med. Chem. 2012, 55 (10), 4872-4876, all of which are herebyincorporated by reference in their entireties.

In some embodiments, a RET inhibitor (e.g., a first RET inhibitor or asecond RET inhibitor) is a compound of the Formula II:

or a pharmaceutically acceptable salt or solvate thereof, wherein:

X¹ is CH, CCH₃, CF, CCl or N;

X² is CH, CF or N;

X³ is CH, CF or N;

X⁴ is CH, CF or N;

wherein zero, one or two of X¹, X², X³ and X⁴ is N;

A is H, Cl, CN, Br, CH₃, CH₂CH₃ or cyclopropyl;

B is hetAr¹;

hetAr¹ is a 5-membered heteroaryl ring having 1-3 ring heteroatomsindependently selected from N, S and O, wherein said heteroaryl ring isoptionally substituted with one or more substituents independentlyselected from the group consisting of halogen, C1-C6 alkyl, hydroxyCl-C6alkyl, fluoroC1-C6 alkyl, difluoroC1-C6 alkyl, trifluoroC1-C6 alkyl,cyanoCl-C6 alkyl, (C1-C6 alkoxy)C1-C6 alkyl, (C1-C4 alkoxy)CH₂C(═O)—,(C1-C4 alkoxy)C(═O)C1-C3 alkyl, C3-C6 cycloalkyl, (R^(a)R^(b)N)C1-C6alkyl, (R^(a)R^(b)N)C(═O)C1-C6 alkyl, (C1-C6 alkyl SO₂)C1-C6 alkyl,hetCyc^(a), and 4-methoxybenzyl;

R^(a) and R^(b) are independently H or C1-C6 alkyl;

hetCyc^(a) is a 4-6 membered heterocyclic ring having a ring heteroatomselected from N and O, wherein said heterocyclic ring is optionallysubstituted with halogen, C1-C6 alkyl, fluoroC1-C6 alkyl, difluoroC1-C6alkyl, trifluoroC1-C6 alkyl, (C1-C6 alkoxy)C1-C6 alkyl, di(C1-C3alkyl)NCH₂C(═O), (C1-C6 alkoxy)C(═O) or (C1-C6 alkoxy)CH₂C(═O);

D is hetCyc¹, hetCyc², hetCyc³ or hetCyc⁹;

hetCyc¹ is a 4-6 membered heterocyclic ring having 1-2 ring atomsselected from N and O, wherein said heterocyclic ring is optionallysubstituted with one or more substituents independently selected fromthe group consisting of C1-C3 alkyl, fluoroC1-C3 alkyl, difluoroC1-C3alkyl, trifluoroC1-C3 alkyl and OH, or said heterocyclic ring issubstituted with a C3-C6 cycloalkylidene ring, or said heterocyclic ringis substituted with an oxo group;

hetCyc² is a 7-8 membered bridged heterocyclic ring having 1-3 ringheteroatoms independently selected from N and O, wherein saidheterocyclic ring is optionally substituted with C1-C3 alkyl;

hetCyc³ is a 7-11 membered heterospirocyclic ring having 1-2 ringheteroatoms independently selected from N and O, wherein said ring isoptionally substituted with C1-C3 alkyl;

hetCyc⁹ is a fused 9-10 membered heterocyclic ring having 1-3 ringnitrogen atoms and optionally substituted with oxo;

E is

-   -   (a) hydrogen,    -   (b) OH,    -   (c) R^(a)R^(b)N—, wherein R^(a) is H or C1-C6 alkyl and R^(b) is        H, C1-C6 alkyl or phenyl;    -   (d) C1-C6 alkyl optionally substituted with one to three        fluoros,    -   (e) hydroxyCl-C6 alkyl- optionally substituted with one to three        fluoros,    -   (f) C1-C6 alkoxy optionally substituted with one to three        fluoros,    -   (g) hydroxy(C1-C6 alkoxy) optionally substituted with one to        three fluoros,    -   (h) (C1-C6 alkoxy)hydroxy C1-C6 alkyl- optionally substituted        with one to three fluoros,    -   (i) (C1-C6 alkyl)C(═O)— optionally substituted with one to three        fluoros,    -   (j) (hydroxy C1-C6 alkyl)C(═O)— optionally substituted with one        to three fluoros,    -   (k) (C1-C6 alkoxy)C(═O)—,    -   (l) (C1-C6 alkoxy)(C1-C6 alkyl)C(═O)—,    -   (m) HC(═O)—,    -   (n) Cyc¹,    -   (o) Cyc¹C(═O)—,    -   (p) Cyc¹(C1-C6 alkyl)C(═O)— wherein said alkyl portion is        optionally substituted with one or more groups independently        selected from the group consisting of OH, fluoro, C1-C3 alkoxy        and R^(c)R^(d)N—, where R^(c) and R^(d) are independently H or        C1-C6 alkyl,    -   (q) hetCyc⁴,    -   (r) hetCyc⁴C(═O)—,    -   (s) hetCyc⁴(C1-C3 alkyl)C(═O)—,    -   (t) (hetCyc⁴)C(═O)C1-C2 alkyl-,    -   (u) hetCyc⁴C(═O)NH—,    -   (v) Ar²,    -   (w) Ar²C(═O)—,    -   (x) Ar²C1-C6 alkyl-,    -   (y) (Ar²)hydroxy C2-C6 alkyl-,    -   (z) Ar²(C1-C3 alkyl)C(═O)— wherein said alkyl portion is        optionally substituted with one or two groups independently        selected from the group consisting of OH, C1-C6 alkyl        (optionally substituted with 1-3 fluoros), hydroxyCl-C6 alkyl,        C1-C6 alkoxy and R^(e)R^(f)N—, where R^(e) and R^(f) are        independently H or C1-C6 alkyl, or R^(e) and R^(f) together with        the nitrogen to which they are attached form a 5-6 membered        azacyclic ring optionally having an additional ring heteroatom        selected from N and O,    -   (aa) hetAr²C(═O)—,    -   (bb) (hetAr²)hydroxyC2-C6 alkyl-,    -   (cc) hetAr²(C1-C3 alkyl)C(═O)—, wherein said alkyl portion is        optionally substituted with one or two groups independently        selected from the group consisting of OH, C1-C6 alkyl,        hydroxyCl-C6 alkyl, C1-C6 alkoxy and R^(e)R^(f)N—, wherein R^(e)        and R^(f) are independently H or C1-C6 alkyl or R^(e) and R^(f)        together with the nitrogen to which they are attached form a 5-6        membered azacyclic ring optionally having an additional ring        heteroatom selected from N and O,    -   (dd) R¹R²NC(═O)—,    -   (ee) R¹R²N(C1-C3 alkyl)C(═O)—, wherein said alkyl portion is        optionally substituted with phenyl,    -   (ff) R¹R²NC(═O)C1-C2 alkyl-,    -   (gg) R¹R²NC(═O)NH—,    -   (hh) CH₃SO₂(C1-C6 alkyl)C(═O)—,    -   (ii) (C1-C6 alkyl)SO₂—,    -   (jj) (C3-C6 cycloalkyl)CH₂SO₂—,    -   (kk) hetCyc⁵-SO₂—,    -   (ll) R⁴R⁵NSO₂—,    -   (mm) R⁶C(═O)NH—,    -   (nn) hetCyc⁶,    -   (oo) hetAr²C1-C6 alkyl-,    -   (pp) (hetCyc⁴)C1-C6 alkyl-,    -   (qq) (C1-C6 alkoxy)C1-C6 alkyl- optionally substituted with 1-3        fluoros,    -   (rr) (C3-C6 cycloalkoxy)C1-C6 alkyl-,    -   (ss) (C3-C6 cycloalkyl)C1-C6 alkyl-, wherein said cycloalkyl is        optionally substituted with 1-2 fluoros,    -   (tt) (R^(g)R^(h)N)C1-C6 alkyl-, wherein R^(g) and R^(h) are        independently H or C1-C6 alkyl,    -   (uu) Ar²—O—,    -   (vv) (C1-C6 alkyl SO₂)C1-C6 alkyl-,    -   (ww) (C1-C6 alkoxy)C(═O)NHC1-C6 alkyl-,    -   (xx) (C3-C6 cycloalkoxy)C(═O)—,    -   (yy) (C3-C6 cycloalkyl)SO₂—, wherein said cycloalkyl is        optionally substituted with C1-C6 alkyl,    -   (zz) Ar⁴CH₂OC(═O)—,    -   (aaa) (N—(C1-C3 alkyl)pyridinonyl)C1-C3 alkyl-, and    -   (bbb) (Ar⁴SO₂)C1-C6 alkyl-;

Cyc¹ is a C3-C6 cycloalkyl, wherein (a) said cycloalkyl is optionallysubstituted with one or more substituents independently selected fromthe group consisting of OH, halogen, C1-C6 alkoxy, CN, hydroxyC1-C6alkyl, (C1-C6 alkoxy)C1-C6 alkyl, and C1-C6 alkyl optionally substitutedwith 1-3 fluoros, or (b) said cycloalkyl is substituted with phenyl,wherein said phenyl is optionally substituted with one or moresubstituents independently selected from the group consisting ofhalogen, C1-C3 alkyl, C1-C3 alkoxy and CF, or (c) said cycloalkyl issubstituted with a 5-6 membered heteroaryl ring having 1-3 ringheteroatoms independently selected from N and O, wherein said heteroarylring is optionally substituted with one or more substituentsindependently selected from the group consisting of halogen, C1-C3alkyl, C1-C3 alkoxy and CF₃;

Ar² is phenyl optionally substituted with one or more substituentsindependently selected from the group consisting of halogen, C1-C6alkyl, C1-C6 alkoxy (optionally substituted with 1-3 fluoros),fluoroC1-C6 alkyl, difluoroC1-C6 alkyl, trifluoroC1-C6 alkyl, CN, a 5-6membered heterocyclic ring having 1-2 ring heteroatoms independentlyselected from N and O, and R^(i)R^(j)N— wherein R^(i) and R^(j) areindependently H or C1-C6 alkyl;

hetAr² is a 5-6 membered heteroaryl ring having 1-3 ring heteroatomsindependently selected from N, O and S and optionally substituted withone or more substituents independently selected from the groupconsisting of halogen, C1-C6 alkyl, C1-C6 alkoxy (optionally substitutedwith 1-3 fluoros), fluoroC1-C6 alkyl, difluoroC1-C6 alkyl,trifluoroC1-C6 alkyl, hydroxyCl-C6 alkyl, (C3-C6)cycloalkyl, (C1-C6alkoxy)C1-C6 alkyl, CN, OH, and R′R″N—, wherein R′ and R″ areindependently H or C1-C3 alkyl;

hetCyc⁴ is (a) a 4-6 membered heterocyclic ring having 1-2 ringheteroatoms independently selected from N, O and S wherein said S isoptionally oxidized to SO₂, (b) a 7-8 membered bridged heterocyclic ringhaving 1-2 ring heteroatoms independently selected from N and O, (c) a6-12 membered fused bicyclic heterocyclic ring having 1-2 ringheteroatoms independently selected from N and O and optionallyindependently substituted with 1-2 C1-C6 alkyl substitutents, or (d) a7-10 membered spirocyclic heterocyclic ring having 1-2 ring heteroatomsindependently selected from N and O, wherein each of said heterocyclicrings is optionally substituted with one or more substituentsindependently selected from the group consisting of halogen, OH, CN,C1-C6 alkyl (optionally substituted with 1-3 fluoros), C1-C6 alkoxy,(C1-C6 alkoxy)C1-C6 alkyl, (C3-C6)cycloalkyl, (C1-C6 alkyl)C(═O)—, a 5-6membered heterocyclic ring having 1-2 ring heteroatoms independentlyselected from N and O, and phenyl wherein said phenyl is optionallysubstituted with one or more substituents selected from halogen, C1-C6alkyl and C1-C6 alkoxy;

hetCyc⁵ is a 5-6 membered heterocyclic ring having a ring heteroatomselected from O and N;

hetCyc⁶ is a 5 membered heterocyclic ring having one or two ringheteroatoms independently selected from N and O, wherein said ring issubstituted with oxo and wherein said ring is further optionallysubstituted with one or more substituents independently selected fromthe group consisting of OH and C1-C6 alkyl;

R¹ is H, C1-C6 alkyl or (C1-C6 alkoxy)C1-C6 alkyl;

R² is H, C1-C6 alkyl (optionally substituted with 1-3 fluoros), (C1-C6alkoxy)C1-C6 alkyl (optionally substituted with 1-3 fluoros), Cyc³,hydroxyCl-C6 alkyl (optionally substituted with 1-3 fluoros), C1-C6alkoxy (optionally substituted with 1-3 fluoros), (C1-C6 alkoxy)C(═O),hetCyc⁷, Ar³, Ar³C1-C3 alkyl-, hydroxyCl-C6 alkoxy or (3-6Ccycloalkyl)CH₂O—;

Cyc³ is a 3-6 membered carbocyclic ring optionally substituted with 1-2groups independently selected from the group consisting of C1-C6 alkoxy,OH and halogen;

hetCyc⁷ is a 5-6 membered heterocyclic ring having a ring heteroatomselected from O and N wherein said ring is optionally substituted withC1-C6 alkyl;

Ar³ is phenyl optionally substituted with one or more substituentsindependently selected from halogen, C1-C3 alkyl, C1-C3 alkoxy,fluoroC1-C3 alkyl, difluoroC1-C3 alkyl and trifluoroC1-C3 alkyl;

R⁴ and R⁵ are independently H or C1-C6 alkyl;

R⁶ is C1-C6 alkyl, hydroxyCl-C6 alkyl, C1-C6 alkoxy, (C1-C6 alkoxy)C1-C6alkyl, phenyl or hetCyc⁸;

hetCyc⁸ is a 5-6 membered heterocyclic ring having a ring heteroatomselected from O and N, wherein said heterocyclic ring is optionallysubstituted with C1-C6 alkyl; and

Ar⁴ is phenyl optionally substituted with one or more halogens.

In some embodiments, a RET inhibitor (e.g., a first RET inhibitor or asecond RET inhibitor) is a compound of the Formula III:

or a pharmaceutically acceptable salt or solvate thereof, wherein:

X¹ is CH or N;

X² is CH or N;

X³ is CH or N;

X⁴ is CH or N;

wherein one or two of X¹, X², X³ and X⁴ is N;

A is CN;

B is hetAr¹;

hetAr¹ is a 5-membered heteroaryl ring having 1-3 ring nitrogen atoms,wherein said heteroaryl ring is optionally substituted with one or moresubstituents independently selected from the group consisting ofhalogen, C1-C6 alkyl, hydroxyC1-C6 alkyl, fluoroC1-C6 alkyl,difluoroC1-C6 alkyl, trifluoroC1-C6 alkyl, cyanoCl-C6 alkyl, (C1-C6alkoxy)C1-C6 alkyl, (C1-C4 alkoxy)CH₂C(═O)—, (C1-C4 alkoxy)C(═O)C1-C3alkyl, C3-C6 cycloalkyl, (R^(a)R^(b)N)C1-C6 alkyl,(R^(a)R^(b)N)C(═O)C1-C6 alkyl, (C1-C6 alkyl SO₂)C1-C6 alkyl, and4-methoxybenzyl;

R^(a) and R^(b) are independently H or C1-C6 alkyl;

D is hetCyc¹;

hetCyc¹ is a 4-6 membered heterocyclic ring having 1-2 ring nitrogenatoms, wherein said heterocyclic ring is optionally substituted with oneor more substituents independently selected from the group consisting ofC1-C3 alkyl, fluoroC1-C3 alkyl, difluoroC1-C3 alkyl, trifluoroC1-C3alkyl and OH, or said heterocyclic ring is substituted with a C3-C6cycloalkylidene ring, or said heterocyclic ring is substituted with anoxo group;

E is

-   -   (w) Ar²C(═O)—,    -   (x) Ar²C1-C6 alkyl-,    -   (z) Ar²(C1-C3 alkyl)C(═O)— wherein said alkyl portion is        optionally substituted with one or two groups independently        selected from the group consisting of OH, C1-C6 alkyl        (optionally substituted with 1-3 fluoros), hydroxyCl-C6 alkyl,        C1-C6 alkoxy and R^(e)R^(f)N—, where R^(e) and R^(f) are        independently H or C1-C6 alkyl, or R^(e) and R^(f) together with        the nitrogen to which they are attached form a 5-6 membered        azacyclic ring optionally having an additional ring heteroatom        selected from N and O,    -   (cc) hetAr²(C1-C3 alkyl)C(═O)—, wherein said alkyl portion is        optionally substituted with one or two groups independently        selected from the group consisting of OH, C1-C6 alkyl,        hydroxyCl-C6 alkyl, C1-C6 alkoxy and R^(e)R^(f)N—, wherein R_(e)        and R^(f) are independently H or C1-C6 alkyl or R_(e) and R^(f)        together with the nitrogen to which they are attached form a 5-6        membered azacyclic ring optionally having an additional ring        heteroatom selected from N and O,    -   (dd) R¹R²NC(═O)—,    -   (oo) hetAr²C1-C6 alkyl-,

Ar² is phenyl optionally substituted with one or more substituentsindependently selected from the group consisting of halogen, C1-C6alkyl, C1-C6 alkoxy (optionally substituted with 1-3 fluoros),fluoroC1-C6 alkyl, difluoroC1-C6 alkyl, trifluoroC1-C6 alkyl, CN, a 5-6membered heterocyclic ring having 1-2 ring heteroatoms independentlyselected from N and O, and R^(i)R^(j)N— wherein R^(i) and R^(j) areindependently H or C1-C6 alkyl;

hetAr² is a 5-6 membered heteroaryl ring having 1-3 ring heteroatomsindependently selected from N, O and S and optionally substituted withone or more substituents independently selected from the groupconsisting of halogen, C1-C6 alkyl, C1-C6 alkoxy (optionally substitutedwith 1-3 fluoros), fluoroC1-C6 alkyl, difluoroC1-C6 alkyl,trifluoroC1-C6 alkyl, hydroxyCl-C6 alkyl, (C3-C6)cycloalkyl, (C1-C6alkoxy)C1-C6 alkyl, CN, OH, and R′R″N—, wherein R′ and R″ areindependently H or C1-C3 alkyl;

R¹ is H, C1-C6 alkyl or (C1-C6 alkoxy)C1-C6 alkyl; and

R² is H, C1-C6 alkyl (optionally substituted with 1-3 fluoros), (C1-C6alkoxy)C1-C6 alkyl (optionally substituted with 1-3 fluoros),hydroxyCl-C6 alkyl (optionally substituted with 1-3 fluoros), C1-C6alkoxy (optionally substituted with 1-3 fluoros), (C1-C6 alkoxy)C(═O),hydroxyCl-C6 alkoxy or (3-6C cycloalkyl)CH₂O.

In some embodiments, a RET inhibitor (e.g., a first RET inhibitor or asecond RET inhibitor) is selected from the group consisting of:(S)-4-(6-(4-(2-hydroxy-3-phenylpropanoyl)piperazin-1-yl)pyridin-3-yl)-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile;6-(1-methyl-1H-pyrazol-4-yl)-4-(6-(4-(2-(pyridin-2-yl)acetyl)piperazin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile;4-(6-(4-(2,6-difluorobenzoyl)piperazin-1-yl)pyridin-3-yl)-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile2,2,2-trifluoroacetate;4-(5-(3-cyano-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-4-yl)pyridin-2-yl)-N,N-diethylpiperazine-1-carboxamide;1-(5-(3-cyano-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-4-yl)pyridin-2-yl)-N-(2-methoxy-3-methylbutyl)piperidine-4-carboxamide;4-(6-(4-(2-(5-fluoropyridin-2-yl)acetyl)piperazin-1-yl)pyridin-3-yl)-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-3-carbonitrilebis(2,2,2-trifluoroacetate);4-(6-(4-(2,6-difluorobenzyl)piperazin-1-yl)pyridine-3-yl)-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile;4-(6-(4-(2-methoxybenzyl)piperazin-1-yl)pyridin-3-yl)-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile;6-(1-methyl-1H-pyrazol-4-yl)-4-(6-(4-(pyridine-2-ylmethyl)piperazin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile;4-(6-(4-((6-methoxypyridin-3-yl)methyl)piperazin-1-yl)pyridin-3-yl)-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile;or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, a RET inhibitor (e.g., a first RET inhibitor or asecond RET inhibitor) is a compound of the Formula IV:

or a pharmaceutically acceptable salt or solvate thereof, wherein:

X¹, X², X³ and X⁴ are independently CH, CF, CCH₃ or N, wherein zero, oneor two of X¹, X², X³ and X⁴ is N;

A is H, CN, Cl, CH₃—, CH₃CH₂—, cyclopropyl, —CH₂CN or —CH(CN)CH₃;

B is

-   -   (a) hydrogen,    -   (b) C1-C6 alkyl optionally substituted with 1-3 fluoros,    -   (c) hydroxyC2-C6 alkyl-, wherein the alkyl portion is optionally        substituted with 1-3 fluoros or a C3-C6 cycloalkylidene ring,    -   (d) dihydroxyC3-C6 alkyl-, wherein the alkyl portion is        optionally substituted with a C3-C6 cycloalkylidene ring,    -   (e) (C1-C6 alkoxy)C1-C6 alkyl- optionally substituted with 1-3        fluoros,    -   (f) (R¹R²N)C1-C6 alkyl- wherein said alkyl portion is optionally        substituted with OH and wherein R¹ and R² are independently H or        C1-C6 alkyl (optionally substituted with 1-3 fluoros);    -   (g) hetAr¹C1-C3 alkyl-, wherein hetAr¹ is a 5-6 membered        heteroaryl ring having 1-3 ring heteroatoms independently        selected from N, O and S and is optionally substituted with one        or more independently selected C1-C6 alkyl substituents;    -   (h) (C3-C6 cycloalkyl)C1-C3 alkyl-, wherein said cycloalkyl is        optionally substituted with OH,    -   (i) (hetCyc^(a))C1-C3 alkyl-,    -   (j) hetCyc^(a)-,    -   (k) C3-C6 cycloalkyl-, wherein said cycloalkyl is optionally        substituted with OH,    -   (l) (C1-C4 alkyl)C(═O)O—C1-C6 alkyl-, wherein each of the C1-C4        alkyl and C1-C6 alkyl portions is optionally and independently        substituted with 1-3 fluoros, or    -   (m) (R¹R²N)C(═O)C1-C6 alkyl-, wherein R^(a) and R² are        independently H or C1-C6 alkyl (optionally substituted with 1-3        fluoros);

hetCyc^(a)- is a 4-6 membered heterocyclic ring having 1-2 ringheteroatoms independently selected from N and O and optionallysubstituted with one or more substituents independently selected fromOH, C1-C6 alkyl (optionally substituted with 1-3 fluoros), hydroxyC1-C6alkyl-, C1-C6 alkoxy, (C1-C6 alkyl)C(═O)—, (C1-C6 alkoxy)C1-C6 alkyl-,and fluoro, or wherein hetCyc^(a) is substituted with oxo;

Ring D is (i) a saturated 4-7 membered heterocyclic ring having two ringnitrogen atoms, (ii) a saturated 7-8 membered bridged heterocyclic ringhaving two ring nitrogen atoms and optionally having a third ringheteroatom which is oxygen, (iii) a saturated 7-11 memberedheterospirocyclic ring having two ring nitrogen atoms, or (iv) asaturated 9-10 membered bicyclic fused heterocyclic ring having two ringnitrogen atoms, wherein each of said rings is optionally substitutedwith (a) one to four groups independently selected from halogen, OH,C1-C3 alkyl which is optionally substituted with 1-3 fluoros, or C1-C3alkoxy which is optionally substituted with 1-3 fluoros, (b) a C3-C6cycloalkylidene ring, or (c) an oxo group;

E is

-   -   (a) hydrogen,    -   (b) C1-C6 alkyl optionally substituted with 1-3 fluoros,    -   (c) (C1-C6 alkoxy)C1-C6 alkyl- optionally substituted with 1-3        fluoros,    -   (d) (C1-C6 alkyl)C(═O)—, wherein said alkyl portion is        optionally substituted with 1-3 fluoros or with a R^(g)R^(h)N—        substituent wherein R^(g) and R^(h) are independently H or C1-C6        alkyl,    -   (e) (hydroxyC2-C6 alkyl)C(═O)— optionally substituted with 1-3        fluoros, (C1-C6 alkoxy)C(═O)—,    -   (g) (C3-C6 cycloalkyl)C(═O)—, wherein said cycloalkyl is        optionally substituted with one or more substituents        independently selected from C1-C6 alkyl, C1-C6 alkoxy, OH, and        (C1-C6 alkoxy)C1-C6 alkyl-, or said cycloalkyl is substituted        with a 5-6 membered heteroaryl ring having 1-3 ring heteroatoms        independently selected from N and O,    -   (h) Ar¹C1-C6 alkyl-,    -   (i) Ar¹(C1-C6 alkyl)C(═O)—, wherein said alkyl portion is        optionally substituted with OH, hydroxyCl-C6 alkyl-, C1-C6        alkoxy, R^(m)R^(n)N— or R^(m)R^(n)N—CH₂—, wherein each R^(m) and        R^(n) is independently H or C1-C6 alkyl,    -   (j) hetAr²C1-C6 alkyl-, wherein said alkyl portion is optionally        substituted with 1-3 fluoros,    -   (k) hetAr²(C1-C6 alkyl)C(═O)— wherein said alkyl portion is        optionally substituted with OH, hydroxyCl-C6 alkyl- or C1-C6        alkoxy,    -   (l) hetAr²C(═O)—,    -   (m) hetCyc¹C(═O)—,    -   (n) hetCyc¹C1-C6 alkyl-,    -   (o) R³R⁴NC(═O)—,    -   (p) Ar¹N(R³)C(═O)—,    -   (q) hetAr²N(R³)C(═O)—,    -   (r) (C1-C6 alkyl)SO₂—, wherein the alkyl portion is optionally        substituted with 1-3 fluoros,    -   (s) Ar¹SO₂—,    -   (t) hetAr²SO₂—,    -   (u) N—(C1-C6 alkyl)pyridinonyl,    -   (v) Ar¹C(═O)—;    -   (w) Ar¹O—C(═O)—,    -   (x) (C3-C6 cycloalkyl)(C1-C6 alkyl)C(═O)—,    -   (y) (C3-C6 cycloalkyl)(C1-C6 alkyl)SO₂—, wherein the alkyl        portion is optionally substituted with 1-3 fluoros,    -   (z) Ar¹(C1-C6 alkyl)SO₂—,    -   (aa) hetCyc¹-O—C(═O)—,    -   (bb) hetCyc¹CH₂C(═O)—,    -   (cc) hetAr², or    -   (dd) C3-C6 cycloalkyl;

Ar¹ is phenyl optionally substituted with one or more substituentsindependently selected from the group consisting of halogen, CN, C1-C6alkyl (optionally substituted with 1-3 fluoros), C1-C6 alkoxy(optionally substituted with 1-3 fluoros), R^(e)R^(f)N— wherein R^(e)and R^(f) are independently H, C1-C6 alkyl, (R^(p)R^(q)N)C1-C6 alkoxy-wherein R^(p) and R^(q) are independently H or C1-C6 alkyl, and(hetAr^(a))C1-C6 alkyl- wherein hetAr^(a) is a 5-6 membered heteroarylring having 1-2 ring nitrogen atoms, or Ar¹ is a phenyl ring fused to a5-6 membered heterocyclic ring having 1-2 ring heteroatoms independentlyselected from N and O;

hetAr² is a 5-6 membered heteroaryl ring having 1-3 ring heteroatomsindependently selected from N, O and S or a 9-10 membered bicyclicheteroaryl ring having 1-3 ring nitrogen atoms, wherein hetAr² isoptionally substituted with one or more substituents independentlyselected from the group consisting of halogen, CN, C1-C6 alkyl(optionally substituted with 1-3 fluoros), C1-C6 alkoxy (optionallysubstituted with 1-3 fluoros), (C1-C6 alkoxy)C1-C6 alkyl- (optionallysubstituted with 1-3 fluoros), R^(e)R^(f)N— wherein R^(e) and R^(f) areindependently H or C1-C6 alkyl, OH, (C1-C6 alkoxy)C1-C6 alkoxy- andC3-C6 cycloalkyl;

hetCyc¹ is a 4-6 membered saturated heterocyclic ring having 1-2 ringheteroatoms independently selected from N, O and S wherein saidheterocyclic ring is optionally substituted with one or moresubstituents independently selected from C1-C6 alkoxy and halogen;

R³ is H or C1-C6 alkyl; and

R⁴ is C1-C6 alkyl.

In some embodiments, a RET inhibitor (e.g., a first RET inhibitor or asecond RET inhibitor) is a compound of the Formula V:

or a pharmaceutically acceptable salt and solvate thereof, wherein:

X¹, X¹, X³ and X⁴ are independently CH or N, wherein zero, one or two ofX¹, X², X³ and X⁴ is N;

A is CN;

B is

-   -   (b) C1-C6 alkyl optionally substituted with 1-3 fluoros,    -   (c) hydroxyC2-C6 alkyl-, wherein the alkyl portion is optionally        substituted with 1-3 fluoros or a C3-C6 cycloalkylidene ring,    -   (e) (C1-C6 alkoxy)C1-C6 alkyl- optionally substituted with 1-3        fluoros,    -   (f) (R¹R²N)C1-C6 alkyl-, wherein said alkyl portion is        optionally substituted with OH and wherein R¹ and R² are        independently H or C1-C6 alkyl (optionally substituted with 1-3        fluoros);    -   (g) hetAr¹C1-C3 alkyl-, wherein hetAr¹ is a 5-6 membered        heteroaryl ring having 1-3 ring heteroatoms independently        selected from N, O and S and is optionally substituted with one        or more independently selected C1-C6 alkyl substituents; or    -   (i) (hetCyc^(a))C1-C3 alkyl-,

hetCyc^(a)- is a 4-6 membered heterocyclic ring having 1-2 ringheteroatoms independently selected from N and O and optionallysubstituted with one or more substituents independently selected fromOH, C1-C6 alkyl (optionally substituted with 1-3 fluoros), hydroxyC1-C6alkyl-, C1-C6 alkoxy, (C1-C6 alkyl)C(═O)—, (C1-C6 alkoxy)C1-C6 alkyl-and fluoro, or wherein hetCyc^(a) is substituted with oxo;

Ring D is (i) a saturated 4-7 membered heterocyclic ring having two ringnitrogen atoms, or (ii) a saturated 7-9 membered bridged heterocyclicring having two ring nitrogen atoms and optionally having a third ringheteroatom which is oxygen, wherein each of said rings is optionallysubstituted with (a) one to four groups independently selected fromhalogen, OH, C1-C3 alkyl which is optionally substituted with 1-3fluoros, or C1-C3 alkoxy which is optionally substituted with 1-3fluoros, (b) a C3-C6 cycloalkylidene ring, or (c) an oxo group;

E is

-   -   (h) Ar¹C1-C6 alkyl-,    -   (j) hetAr²C1-C6 alkyl-, wherein the alkyl portion is optionally        substituted with 1-3 fluoros, or    -   (l) hetAr²C(═O)—,

Ar¹ is phenyl optionally substituted with one or more substituentsindependently selected from the group consisting of halogen, CN, C1-C6alkyl (optionally substituted with 1-3 fluoros), C1-C6 alkoxy(optionally substituted with 1-3 fluoros), R^(e)R^(f)N— wherein R^(e)and R^(f) are independently H or C1-C6 alkyl, (R^(p)R^(q)N)C1-C6 alkoxy-wherein R^(p) and R^(q) are independently H or C1-C6 alkyl, and(hetAr^(a))C1-C6 alkyl- wherein hetAr^(a) is a 5-6 membered heteroarylring having 1-2 ring nitrogen atoms, or Ar¹ is a phenyl ring fused to a5-6 membered heterocyclic ring having 1-2 ring heteroatoms independentlyselected from N and O; and

hetAr² is a 5-6 membered heteroaryl ring having 1-3 ring heteroatomsindependently selected from N, O and S or a 9-10 membered bicyclicheteroaryl ring having 1-3 ring nitrogen atoms, wherein hetAr² isoptionally substituted with one or more substituents independentlyselected from the group consisting of halogen, CN, C1-C6 alkyl(optionally substituted with 1-3 fluoros), C1-C6 alkoxy (optionallysubstituted with 1-3 fluoros), (C1-C6 alkoxy)C1-C6 alkyl- (optionallysubstituted with 1-3 fluoros), R^(e)R^(f)N— wherein R^(e) and R^(f) areindependently H or C1-C6 alkyl, OH, (C1-C6 alkoxy)C1-C6 alkoxy- andC3-C6 cycloalkyl.

In some embodiments, a RET inhibitor (e.g., a first RET inhibitor or asecond RET inhibitor) is selected from the group consisting of:4-(6-(4-benzylpiperazin-1-yl)pyridin-3-yl)-6-(2-morpholinoethoxy)pyrazolo[1,5-a]pyridine-3-carbonitrile;6-(2-hydroxyethoxy)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile;(R)-6-(2-hydroxypropoxy)-4-(6-(4-((6-methoxypyridin-3-yl)methyl)piperazin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile;6-(2-hydroxy-2-methylpropoxy)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile;6-(2-methoxyethoxy)-4-(6-(4-((6-methoxypyridin-3-yl)methyl)piperazin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile;6-(2-hydroxy-2-methylpropoxy)-4-(6-(6-(6-methoxynicotinoyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile;6-(2-(dimethylamino)ethoxy)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile;4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)-6-(2-morpholinoethoxy)pyrazolo[1,5-a]pyridine-3-carbonitrile;4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)-6-((1-methyl-1H-imidazol-4-yl)methoxy)pyrazolo[1,5-a]pyridine-3-carbonitrile;and6-ethoxy-4-(5-(6-((5-fluoro-6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyrazin-2-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile;or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, a RET inhibitor (e.g., a first RET inhibitor or asecond RET inhibitor) is a compound of Formula VI:

or a pharmaceutically acceptable salt or solvate thereof, wherein:

X¹, X², X³ and X⁴ are independently CH, CCH₃, CF or N, wherein zero, oneor two of X¹, X², X³ and X⁴ is N;

A is H, CN, Cl, methyl, ethyl or cyclopropyl;

B is:

-   -   (a) hydrogen,    -   (b) C1-C6 alkyl optionally substituted with 1-3 fluoros,    -   (c) hydroxyC2-C6 alkyl- wherein the alkyl portion is optionally        substituted with a C3-C6 cycloalkylidene ring,    -   (d) dihydroxyC3-C6 alkyl- wherein the alkyl portion is        optionally substituted with a C3-C6 cycloalkylidene ring,    -   (e) (C1-C6 alkoxy)C1-C6 alkyl- optionally substituted with 1-3        fluoros,    -   (f) (R¹R²N)C1-C6 alkyl- where R¹ and R² are independently        selected from H, C1-C6 alkyl (optionally substituted with 1-3        fluoros), (C1-C6 alkoxy)C1-C6 alkyl-, (C1-C6 alkyl)C(═O)— and        (C1-C6 alkoxy)C(═O)—;    -   (g) hetAr¹C1-C3 alkyl-, where hetAr¹ is a 5-6 membered        heteroaryl ring having 1-3 ring heteroatoms independently        selected from N, O and S and is optionally substituted with one        or more independently selected C1-C6 alkyl substituents;    -   (h) (C3-C6 cycloalkyl)C1-C3 alkyl-, wherein said cycloalkyl is        optionally substituted with OH,    -   (i) (hetCyc^(a))C1-C3 alkyl-,    -   (j) hetCyc^(a),    -   (k) (R¹R²N)C(═O)C1-C6 alkyl-, where R¹ and R² are independently        selected from H and C1-C6 alkyl;    -   (l) (R¹R²N)C(═O)—, where R¹ and R² are independently selected        from H and C1-C6 alkyl, or    -   (m) hetCyc^(a)C(═O)C1-C6 alkyl-;

hetCyc^(a) is a 4-6 membered heterocyclic ring having 1-2 ringheteroatoms independently selected from N and O and optionallysubstituted with one or more substituents independently selected fromOH, C1-C6 alkyl (optionally substituted with 1-3 fluoros), hydroxyCl-C6alkyl, halogen, (C1-C6 alkyl)C(═O)—, C1-C6 alkoxy, oxo and (C1-C6alkoxy)C(═O)—;

Ring D is (i) a saturated monocyclic 4-7 membered heterocyclic ringhaving one ring heteroatom which is nitrogen, (ii) a saturated 7-8membered bridged heterocyclic ring having one ring heteroatom which isnitrogen, or (iii) a saturated 7-11 membered heterospirocyclic ringsystem having one ring heteroatom which is nitrogen;

each R^(a) is independently C1-C6 alkyl (optionally substituted with 1-3fluoros), hydroxyCl-C6 alkyl or (C1-C6 alkoxy)C1-C6 alkyl-;

R^(b) is (a) hydroxy, (b) cyclopropyl, (c) hetCyc^(b)CH₂—, (d)R^(i)R^(j)NC(═O)CH₂OCH₂— where R^(i) and R^(j) are independently H orC1-C6 alkyl, (e) R^(c)R^(d)N—, (f) R^(c)R^(d)NCH₂—, (g) C1-C6 alkoxy-,(h) (C1-C4 alkyl)-C(═O)NH— wherein said alkyl portion is optionallysubstituted with hetCyc^(b), hetAr^(a), C1-C6 alkoxy- or R′R″N—, or saidalkyl portion is optionally substituted with two substituentsindependently selected from R′R″N— and OH, where each R′ and R″ isindependently hydrogen or C1-C6 alkyl, (i) (R¹R″N)C1-C6 alkoxy(CH₂)_(n)—where n is 0 or 1 and R′ and R″ are independently hydrogen or C1-C6alkyl, (j) hetCyc^(b)(C1-C3 alkyl)OCH₂—, (k) hetCyc^(b)C(═O)NH— or (l)hetAr^(a)C(═O)NH—;

hetCyc^(b) is a 4-6 membered heterocyclic ring, a 7-8 membered bridgedheterocyclic ring, or a 7-10 membered heterospirocyclic ring, each ringhaving 1-2 ring heteroatoms independently selected from N and O, whereinhetCyc^(b) is optionally substituted with one or more substituentsindependently selected from OH, fluoro, C1-C6 alkyl (optionallysubstituted with 1-3 fluoros), hydroxyCl-C6 alkyl- (optionallysubstituted with 1-3 fluoros), (C1-C6 alkoxy)C1-C6 alkyl-, (C1-C6alkoxy)C(═O)—, C1-C6 alkoxy, and R¹R″N— where R′ and R″ areindependently hydrogen or C1-C6 alkyl;

hetAr^(a) is a 5-6 membered heteroaryl ring having 1-3 ring heteroatomsindependently selected from N, O and S wherein hetAr^(a) is optionallysubstituted with one or more substituents independently selected fromthe group consisting of halogen, CN, C1-C6 alkyl (optionally substitutedwith 1-3 fluoros), and C1-C6 alkoxy (optionally substituted with 1-3fluoros),

R^(c) is hydrogen or C1-C6 alkyl;

R^(d) is hydrogen, C1-C6 alkyl (optionally substituted with 1-3fluoros), (C1-C6 alkoxy)C(═O)—, hydroxyC1-C6 alkyl (optionallysubstituted with 1-3 fluoros), (hydroxyCl-C6 alkyl)C(═O)—, (C1-C6alkyl)C(═O)—, (R^(k)R^(l)N)C1-C6 alkyl- where R^(k) and R^(l) areindependently H or C1-C6 alkyl, R^(m)R^(n)NC(═O)C1-C6 alkyl- where R^(m)and R^(n) are independently H or C1-C6 alkyl, PhCH₂— wherein the phenylis optionally substituted with one or more substituents independentlyselected from the group consisting of halogen, CN, C1-C6 alkyl(optionally substituted with 1-3 fluoros), C1-C6 alkoxy (optionallysubstituted with 1-3 fluoros), (C1-C6 alkoxy)C1-C6 alkyl- (optionallysubstituted with 1-3 fluoros), C3-C6 cycloalkyl, hydroxyCl-C6 alkyl,(C1-C6 alkyl)SO₂—, R^(e)R^(f)N— and (R^(e)R^(f)N)C1-C6 alkyl- where eachR^(e) and R^(f) is independently H or C1-C6 alkyl, (C1-C6 alkoxy)C1-C6alkyl-, or hetCyc^(c) where hetCyc^(c) is a 4-6 membered heterocyclicring having a ring heteroatom selected from N and O and optionallysubstituted with C1-C6 alkyl;

n is 0, 1, 2, 3, 4, 5 or 6;

m is 0 or 1;

E is:

-   -   (a) hydrogen,    -   (b) hydroxy,    -   (c) C1-C6 alkyl optionally substituted with 1-3 fluoros,    -   (d) Ar¹C1-C6 alkyl- wherein said alkyl portion is optionally        substituted with 1-3 fluoros,    -   (e) hetAr²C1-C6 alkyl-,    -   (f) (C1-C6 alkoxy)C1-C6 alkoxy-,    -   (g) Ar¹O—,    -   (h) hetAr²—O—,    -   (i) Ar¹NR^(g)— where R^(g) is H or C1-C6 alkyl,    -   (j) hetAr²NR^(g)— where R^(g) is H or C1-C6 alkyl,    -   (k) R³C(═O)NR^(g)— where R^(g) is H or C1-C6 alkyl;    -   (l) Ar¹C(═O)NR^(g)— where R^(g) is H or C1-C6 alkyl,    -   (m) hetAr²C(═O)NR^(g)(CH₂)_(p)— where p is 0 or 1 and R^(g) is H        or C1-C6 alkyl,    -   (n) R⁴R⁵NC(═O)—,    -   (o) Ar¹NR^(g)C(═O)—, where R^(g) is H or C1-C6 alkyl,    -   (p) hetAr²NR^(g)C(═O)—, where R^(g) is H or C1-C6 alkyl,    -   (q) Ar¹(C1-C6 alkyl)C(═O)— wherein said alkyl portion is        optionally substituted with OH, hydroxy(C1-C6 alkyl), C1-C6        alkoxy or NH₂,    -   (r) hetCyc⁵C(═O)—,    -   (s) R⁴R⁵NC(═O)NR^(g)— where R^(g) is H or C1-C6 alkyl, or    -   (t) (C1-C6 alkyl)SO₂—;    -   (u) Ar¹(C1-C6 alkyl)C(═O)NR^(g)— where R^(g) is H or C1-C6        alkyl,    -   (v) hetAr⁴C(═O)NR^(g)— where R^(g) is H or C1-C6 alkyl,    -   (w) hetAr²—S(═O)—,    -   (x) (C3-C6 cycloalkyl)CH₂SO₂—,    -   (y) Ar¹(C1-C6 alkyl)SO₂—,    -   (z) hetAr²SO₂—,    -   (aa) Ar¹,    -   (bb) hetAr²,    -   (cc) hetCyc⁵,    -   (dd) C1-C6 alkoxy,    -   (ee) Ar¹(C1-C6 alkyl)-O—,    -   (ff) hetAr²(C1-C6 alkyl)-O—,    -   (gg) hetAr²—O—C1-C6 alkyl-,    -   (hh) Ar¹(C1-C6 alkyl)NR^(g)— where R^(g) is H or C1-C6 alkyl,    -   (ii) hetAr²—S—,    -   (jj) Ar²SO₂NR^(g)(CH₂)_(p)— where p is 0 or 1 and R^(g) is H or        C1-C6 alkyl,    -   (kk) (C1-C6 alkoxy)C(═O)—,    -   (ll) (C1-C6 alkyl)NR^(g)C(═O)O— where R^(g) is H or C1-C6 alkyl,    -   (mm) (C1-C6 alkyl)NR^(g)SO₂— where R^(g) is H or C1-C6 alkyl,    -   (nn) hetCyc⁵C(═O)NR^(g)— where R^(g) is H or C1-C6 alkyl,    -   (oo) Q-NR^(h)(C1-C3 alkyl)C(═O)NR^(g)— where R^(g) and R^(h) are        independently H or C1-C6 alkyl and Q is H, C1-C6 alkyl or (C1-C6        alkyl)OC(═O)—,    -   (pp)

-   -    where R^(g) and R^(h) are independently H or C1-C6 alkyl, Q is        H, C1-C6 alkyl or (C1-C6 alkyl)OC(═O)— and r is 1, 2, 3 or 4,    -   (qq)

-   -    where R^(g) and R^(h) are independently H or C1-C6 alkyl and Q        is H, C1-C6 alkyl or (C1-C6 alkyl)OC(═O)—,    -   (rr)

-   -    where R^(g) is H or C1-C6 alkyl and Q is H, C1-C6 alkyl or        (C1-C6 alkyl)OC(═O)—, or    -   (ss) R^(g)R^(h)N— where R^(g) and R^(h) are independently H or        C1-C6 alkyl,    -   (tt) (C3-C6 cycloalkyl)C(═O)NR^(g)— where the cycloalkyl is        optionally and independently substituted with one or more        halogens,    -   (uu) (C1-C6 alkyl)C(═O)NR^(g)CH₂— where R^(g) is H or C1-C6        alkyl, or    -   (vv) C1-C6 alkyl)SO₂NR^(g)— where R^(g) is H or C1-C6 alkyl;

Ar¹ is phenyl optionally substituted with one or more substituentsindependently selected from the group consisting of halogen, CN, C1-C6alkyl (optionally substituted with 1-3 fluoros), C1-C6 alkoxy(optionally substituted with 1-3 fluoros), (C1-C6 alkoxy)C1-C6 alkyl-(optionally substituted with 1-3 fluoros), C3-C6 cycloalkyl,hydroxyCl-C6 alkyl, (C1-C6 alkyl)SO₂—, R^(e)R^(f)N— and(R^(e)R^(f)N)C1-C6 alkyl- where each R^(e) and R^(f) is independently Hor C1-C6 alkyl;

hetAr² is a 5-6 membered heteroaryl ring having 1-3 ring heteroatomsindependently selected from N, O and S, or a 9-10 membered bicyclicheteroaryl having 1-2 ring nitrogen atoms, wherein hetAr² is optionallysubstituted with one or more substituents independently selected fromthe group consisting of halogen, CN, C1-C6 alkyl (optionally substitutedwith 1-3 fluoros), C1-C6 alkoxy (optionally substituted with 1-3fluoros), (C1-C6 alkoxy)C1-C6 alkyl- (optionally substituted with 1-3fluoros) and hydroxyCl-C6 alkoxy-;

hetCyc⁵ is a 4-6 membered saturated heterocyclic ring having 1-2 ringheteroatoms independently selected from N, O and S wherein saidheterocyclic ring is optionally substituted with one or moresubstituents independently selected from C1-C6 alkoxy and oxo;

R³ is C1-C6 alkyl (optionally substituted with 1-3 fluoros),hydroxyCl-C6 alkyl-, C1-C6 alkoxy, C3-C6 cycloalkyl, (C3-C6cycloalkyl)CH₂—, (C3-C6 cycloalkyl)O—, (C3-C6 cycloalkyl)CH₂O—,hetCyc⁷O—, Ph-O—, or (C1-C6 alkoxy)C1-C6 alkyl-; wherein each of saidC3-C6 cycloalkyl moieties is optionally substituted with C1-C6 alkyl(optionally substituted with 1-3 fluoros), C1-C6 alkoxy, OH or R′R″N—where R′ and R″ are independently hydrogen or C1-C6 alkyl;

R⁴ is H or C1-C6 alkyl;

R⁵ is Ar², hetAr³, Ar²CH₂—, hetCyc⁶-CH₂—, hydroxyCl-C6 alkyl-, (C3-C6cycloalkyl)CH₂—, or C1-C6 alkyl optionally substituted with 1-3 fluoros;

Ar² is phenyl optionally substituted with one or more substituentsindependently selected from the group consisting of halogen, CN, C1-C6alkyl (optionally substituted with 1-3 fluoros), C1-C6 alkoxy(optionally substituted with 1-3 fluoros), (C1-C6 alkoxy)C1-C6 alkyl-(optionally substituted with 1-3 fluoros), C3-C6 cycloalkyl, andR^(g)R^(h)N— where R^(g) and R^(h) are independently H or C1-C6 alkyl,or Ar² is phenyl fused to a 6 membered heterocyclic ring having a ringnitrogen atom and optionally substituted with C1-C6 alkyl;

hetAr³ is a 5-6 membered heteroaryl ring having 1-3 ring heteroatomsindependently selected from N, O and S and optionally substituted withone or more substituents independently selected from the groupconsisting of halogen, CN, C1-C6 alkyl (optionally substituted with 1-3fluoros), C1-C6 alkoxy (optionally substituted with 1-3 fluoros), and(C1-C6 alkoxy)C1-C6 alkyl-(optionally substituted with 1-3 fluoros);

hetAr⁴ is pyridin-4(1H)-onyl or pyridin-2(1H)-onyl optionallysubstituted with one or more substituents independently selected fromC1-C6 alkyl and halogen;

hetCyc⁶ is a 5-7 membered heterocyclic ring having 1-3 ring heteroatomsindependently selected from N, O and S; and

hetCyc⁷ is a 5-7 membered heterocyclic ring having 1-3 ring heteroatomsindependently selected from N, O and S.

In some embodiments, a RET inhibitor (e.g., a first RET inhibitor or asecond RET inhibitor) is a compound of the Formula VII:

or a pharmaceutically acceptable salt or solvate thereof, wherein:

X³ and X⁴ are independently CH or N, wherein zero, one or two of X¹, X²,X³ and X⁴ is N;

A is CN;

B is:

-   -   (b) C1-C6 alkyl optionally substituted with 1-3 fluoros,    -   (c) hydroxyC2-C6 alkyl- wherein the alkyl portion is optionally        substituted with a C3-C6 cycloalkylidene ring, or    -   (i) (hetCyc^(a))C1-C3 alkyl-;    -   hetCyc^(a) is a 4-6 membered heterocyclic ring having 1-2 ring        heteroatoms independently selected from N and O and optionally        substituted with one or more substituents independently selected        from OH, C1-C6 alkyl (optionally substituted with 1-3 fluoros),        hydroxyCl-C6 alkyl, halogen, (C1-C6 alkyl)C(═O)—, C1-C6 alkoxy,        oxo, and (C1-C6 alkoxy)C(═O)—;

Ring D is a saturated monocyclic 4-7 membered heterocyclic ring havingone ring heteroatom which is nitrogen;

each R^(a) is independently C1-C6 alkyl (optionally substituted with 1-3fluoros);

R^(b) is (a) hydroxy;

n is 0 or 1;

m is 0 or 1;

E is:

-   -   (e) hetAr²C1-C6 alkyl-,    -   (h) hetAr²—O—,    -   (k) R³C(═O)NR^(g)— where R^(g) is H or C1-C6 alkyl,    -   (l) Ar¹C(═O)NR^(g)— where R^(g) is H or C1-C6 alkyl, or    -   (m) hetAr²C(═O)NR^(g)(CH₂)_(p)— where p is 0 or 1 and R^(g) is H        or C1-C6 alkyl;

Ar¹ is phenyl optionally substituted with one or more substituentsindependently selected from the group consisting of halogen, CN, C1-C6alkyl (optionally substituted with 1-3 fluoros), C1-C6 alkoxy(optionally substituted with 1-3 fluoros), (C1-C6 alkoxy)C1-C6 alkyl-(optionally substituted with 1-3 fluoros), C3-C6 cycloalkyl,hydroxyCl-C6 alkyl, (C1-C6 alkyl)SO₂—, R^(e)R^(f)N— and(R^(e)R^(f)N)C1-C6 alkyl- where each R^(e) and R^(f) is independently Hor C1-C6 alkyl;

hetAr² is a 5-6 membered heteroaryl ring having 1-3 ring heteroatomsindependently selected from N, O and S, or a 9-10 membered bicyclicheteroaryl having 1-2 ring nitrogen atoms, wherein hetAr² is optionallysubstituted with one or more substituents independently selected fromthe group consisting of halogen, CN, C1-C6 alkyl (optionally substitutedwith 1-3 fluoros), C1-C6 alkoxy (optionally substituted with 1-3fluoros), (C1-C6 alkoxy)C1-C6 alkyl- (optionally substituted with 1-3fluoros) and hydroxyCl-C6 alkoxy-; and

R³ is C1-C6 alkyl (optionally substituted with 1-3 fluoros),hydroxyCl-C6 alkyl-, C1-C6 alkoxy, C3-C6 cycloalkyl, (C3-C6cycloalkyl)CH₂—, (C3-C6 cycloalkyl)O—, (C3-C6 cycloalkyl)CH₂O—,hetCyc⁷O—, Ph-O—, or (C1-C6 alkoxy)C1-C6 alkyl-; wherein each of saidC3-C6 cycloalkyl moieties is optionally substituted with C1-C6 alkyl(optionally substituted with 1-3 fluoros), C1-C6 alkoxy, OH, or R′R″N—where R′ and R″ are independently hydrogen or C1-C6 alkyl.

In some embodiments, a RET inhibitor (e.g., a first RET inhibitor or asecond RET inhibitor) is selected from the group consisting of:N-(1-(5-(3-cyano-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridin-4-yl)pyridin-2-yl)-4-methylpiperidin-4-yl)benzamide;6-ethoxy-4-(6-(4-hydroxy-4-(pyridin-2-ylmethyl)piperidin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile;6-(2-hydroxy-2-methylpropoxy)-4-(6-(3-(pyridin-2-yloxy)azetidin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile;6-(2-hydroxy-2-methylpropoxy)-4-(6-(4-((6-methoxypyridazin-3-yl)oxy)piperidin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile;(S)-6-(2-hydroxy-2-methylpropoxy)-4-(6-(3-(pyridin-2-yloxy)pyrrolidin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile;N-(1-(5-(3-cyano-6-((3-fluoro-1-methylazetidin-3-yl)methoxy)pyrazolo[1,5-a]pyridin-4-yl)pyridin-2-yl)-4-methylpiperidin-4-yl)-5-fluoro-2-methylbenzamide;3-chloro-N-(1-(5-(3-cyano-6-((3-fluoro-1-methylazetidin-3-yl)methoxy)pyrazolo[1,5-a]pyridin-4-yl)pyridin-2-yl)-4-methylpiperidin-4-yl)picolinamide;N-((3S,4S)-1-(5-(3-cyano-6-ethoxypyrazolo[1,5-a]pyridin-4-yl)pyridin-2-yl)-3-hydroxypiperidin-4-yl)-3-methylbutanamide;6-(2-hydroxy-2-methylpropoxy)-4-(6-(4-hydroxy-4-(pyridin-2-ylmethyl)piperidin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile;and 3-chloro-N-((3S,4S)-1-(5-(3-cyano-6-ethoxypyrazolo[1,5-a]pyridin-4-yl)pyrazin-2-yl)-3-hydroxypiperidin-4-yl)picolinamide;or a pharmaceutically acceptable salt or solvate thereof.

Non-limiting examples of receptor tyrosine kinase (e.g., Trk) targetedtherapeutic agents, include afatinib, cabozantinib, cetuximab,crizotinib, dabrafenib, entrectinib, erlotinib, gefitinib, imatinib,lapatinib, lestaurtinib, nilotinib, pazopanib, panitumumab, pertuzumab,sunitinib, trastuzumab, 1-((3S,4R)-4-(3-fluorophenyl)-1-(2-methoxyethyl)pyrrolidin-3-yl)-3-(4-methyl-3-(2-methylpyrimidin-5-yl)-1-phenyl-1H-pyrazol-5-yl)urea,AG 879, AR-772, AR-786, AR-256, AR-618, AZ-23, AZ623, DS-6051, Gö 6976,GNF-5837, GTx-186, GW 441756, LOXO-101, MGCD516, PLX7486, RXDX101,VM-902A, TPX-0005, and TSR-011. Additional Trk targeted therapeuticagents include those described in U.S. Pat. Nos. 8,450,322; 8,513,263;8,933,084; 8,791,123; 8,946,226; 8,450,322; 8,299,057; and 8,912,194;U.S. Publication No. 2016/0137654; 2015/0166564; 2015/0051222;2015/0283132; and 2015/0306086; International Publication No. WO2010/033941; WO 2010/048314; WO 2016/077841; WO 2011/146336; WO2011/006074; WO 2010/033941; WO 2012/158413; WO 2014078454; WO2014078417; WO 2014078408; WO 2014078378; WO 2014078372; WO 2014078331;WO 2014078328; WO 2014078325; WO 2014078323; WO 2014078322; WO2015175788; WO 2009/013126; WO 2013/174876; WO 2015/124697; WO2010/058006; WO 2015/017533; WO 2015/112806; WO 2013/183578; and WO2013/074518, all of which are hereby incorporated by reference in theirentireties.

Further examples of Trk inhibitors can be found in U.S. Pat. No.8,637,516, International Publication No. WO 2012/034091, U.S. Pat. No.9,102,671, International Publication No. WO 2012/116217, U.S.Publication No. 2010/0297115, International Publication No. WO2009/053442, U.S. Pat. No. 8,642,035, International Publication No. WO2009092049, U.S. Pat. No. 8,691,221, International Publication No.WO2006131952, all of which are incorporated by reference in theirentireties herein. Exemplary Trk inhibitors include GNF-4256, describedin Cancer Chemother. Pharmacol. 75(1):131-141, 2015; and GNF-5837(N-[3-[[2,3-dihydro-2-oxo-3-(1H-pyrrol-2-ylmethylene)-1H-indol-6-yl]amino]-4-methylphenyl]-N′-[2-fluoro-5-(trifluoromethyl)phenyl]-urea),described in ACS Med. Chem. Lett. 3(2):140-145, 2012, each of which isincorporated by reference in its entirety herein.

Additional examples of Trk inhibitors include those disclosed in U.S.Publication No. 2010/0152219, U.S. Pat. No. 8,114,989, and InternationalPublication No. WO 2006/123113, all of which are incorporated byreference in their entireties herein. Exemplary Trk inhibitors includeAZ623, described in Cancer 117(6):1321-1391, 2011; AZD6918, described inCancer Biol. Ther. 16(3):477-483, 2015; AZ64, described in CancerChemother. Pharmacol. 70:477-486, 2012; AZ-23((S)-5-Chloro-N2-(1-(5-fluoropyridin-2-yl)ethyl)-N4-(5-isopropoxy-1H-pyrazol-3-yl)pyrimidine-2,4-diamine),described in Mol. Cancer Ther. 8:1818-1827, 2009; and AZD7451; each ofwhich is incorporated by reference in its entirety.

A Trk inhibitor can include those described in U.S. Pat. Nos. 7,615,383;7,384,632; 6,153,189; 6,027,927; 6,025,166; 5,910,574; 5,877,016; and5,844,092, each of which is incorporated by reference in its entirety.

Further examples of Trk inhibitors include CEP-751, described in Int. J.Cancer 72:672-679, 1997; CT327, described in Acta Derm. Venereol.95:542-548, 2015; compounds described in International Publication No.WO 2012/034095; compounds described in U.S. Pat. No. 8,673,347 andInternational Publication No. WO 2007/022999; compounds described inU.S. Pat. No. 8,338,417; compounds described in InternationalPublication No. WO 2016/027754; compounds described in U.S. Pat. No.9,242,977; compounds described in U.S. Publication No. 2016/0000783;sunitinib(N-(2-diethylaminoethyl)-5-[(Z)-(5-fluoro-2-oxo-1H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide),as described in PLoS One 9: e95628, 2014; compounds described inInternational Publication No. WO 2011/133637; compounds described inU.S. Pat. No. 8,637,256; compounds described in Expert. Opin. Ther. Pat.24(7):731-744, 2014; compounds described in Expert Opin. Ther. Pat.19(3):305-319, 2009; (R)-2-phenylpyrrolidine substitutedimidazopyridazines, e.g., GNF-8625,(R)-1-(6-(6-(2-(3-fluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-[2,4′-bipyridin]-2′-yl)piperidin-4-olas described in ACS Med. Chem. Lett. 6(5):562-567, 2015; GTx-186 andothers, as described in PLoS One 8(12):e83380, 2013; K₂₅₂a((9S-(9α,10β,12α))-2,3,9,10,11,12-hexahydro-10-hydroxy-10-(methoxycarbonyl)-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-k1]pyrrolo[3,4-i][1,6]benzodiazocin-1-one),as described in Mol. Cell Biochem. 339(1-2):201-213, 2010;4-aminopyrazolylpyrimidines, e.g., AZ-23(((S)-5-chloro-N2-(1-(5-fluoropyridin-2-yl)ethyl)-N4-(5-isopropoxy-1H-pyrazol-3-yl)pyrimidine-2,4-diamine)),as described in J. Med. Chem. 51(15):4672-4684, 2008; PHA-739358(danusertib), as described in Mol. Cancer Ther. 6:3158, 2007; Gö 6976(5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propanenitrile),as described in J. Neurochem. 72:919-924, 1999; GW441756((3Z)-3-[(1-methylindol-3-yl)methylidene]-1H-pyrrolo[3,2-b]pyridin-2-one),as described in IJAE 115:117, 2010; milciclib (PHA-848125AC), describedin J. Carcinog. 12:22, 2013; AG-879((2E)-3-[3,5-Bis(1,1-dimethylethyl)-4-hydroxyphenyl]-2-cyano-2-propenethioamide);altiratinib(N-(4-((2-(cyclopropanecarboxamido)pyridin-4-yl)oxy)-2,5-difluorophenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide);cabozantinib(N-(4-((6,7-Dimethoxyquinolin-4-yl)oxy)phenyl)-N′-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide);lestaurtinib ((5 S,6 S,8R)-6-Hydroxy-6-(hydroxymethyl)-5-methyl-7,8,14,15-tetrahydro-5H-16-oxa-4b,8a,14-triaza-5,8-methanodibenzo[b,h]cycloocta[jkl]cyclopenta[e]-as-indacen-13(6H)-one);dovatinib(4-amino-5-fluoro-3-[6-(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl]quinolin-2(1H)-onemono 2-hydroxypropanoate hydrate); sitravatinib(N-(3-fluoro-4-((2-(5-(((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide);ONO-5390556; regorafenib(4-[4-({[4-Chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)-3-fluorophenoxy]-N-methylpyridine-2-carboxamidehydrate); and VSR-902A; all of the references above are incorporated byreference in their entireties herein.

The ability of a Trk inhibitor to act as a TrkA, TrkB, and/or Trk Cinhibitor may be tested using the assays described in Examples A and Bin U.S. Pat. No. 8,513,263, which is incorporated herein by reference.

In some embodiments, the receptor tyrosine kinase inhibitor is anepidermal growth factor receptor typrosine kinase inhibitor (EGFR). Forexample, EGFR inhibitors can include osimertinib (merelectinib,Tagrisso), erlotinib (Tarceva), gefitinib (Iressa), cetuximab (Erbitux),necitumumab (Portrazza), neratinib (Nerlynx), lapatinib (Tykerb),panitumumab (Vectibix), and vandetanib (Caprelsa). In some embodiments,the EGFR inhibitor is osimertinib.

In some embodiments, signal transduction pathway inhibitors includeRas-Raf-MEK-ERK pathway inhibitors (e.g., binimetinib, selumetinib,encorafinib, sorafenib, trametinib, and vemurafenib), PI3K-Akt-mTOR-S6Kpathway inhibitors (e.g. everolimus, rapamycin, perifosine,temsirolimus), and other kinase inhibitors, such as baricitinib,brigatinib, capmatinib, danusertib, ibrutinib, milciclib, quercetin,regorafenib, ruxolitinib, semaxanib, AP32788, BLU285, BLU554, INCB39110,INCB40093, INCB50465, INCB52793, INCB54828, MGCD265, NMS-088,NMS-1286937, PF 477736((R)-amino-N-[5,6-dihydro-2-(1-methyl-1H-pyrazol-4-yl)-6-oxo-1Hpyrrolo[4,3,2-ef][2,3]benzodiazepin-8-yl]-cyclohexaneacetamide),PLX3397, PLX7486, PLX8394, PLX9486, PRN1008, PRN1371, RXDX103, RXDX106,RXDX108, and TG101209(N-tert-butyl-3-(5-methyl-2-(4-(4-methylpiperazin-1-yl)phenylamino)pyrimidin-4-ylamino)benzenesulfonamide).

Non-limiting examples of checkpoint inhibitors include ipilimumab,tremelimumab, nivolumab, pidilizumab, MPDL3208A, MEDI4736, MSB0010718C,BMS-936559, BMS-956559, BMS-935559 (MDX-1105), AMP-224, andpembrolizumab.

In some embodiments, cytotoxic chemotherapeutics are selected fromarsenic trioxide, bleomycin, cabazitaxel, capecitabine, carboplatin,cisplatin, cyclophosphamide, cytarabine, dacarbazine, daunorubicin,docetaxel, doxorubicin, etoposide, fluorouracil, gemcitabine,irinotecan, lomustine, methotrexate, mitomycin C, oxaliplatin,paclitaxel, pemetrexed, temozolomide, and vincristine.

Non-limiting examples of angiogenesis-targeted therapies includeaflibercept and bevacizumab.

The term “immunotherapy” refers to an agent that modulates the immunesystem. In some embodiments, an immunotherapy can increase theexpression and/or activity of a regulator of the immune system. In someembodiments, an immunotherapy can decrease the expression and/oractivity of a regulator of the immune system. In some embodiments, animmunotherapy can recruit and/or enhance the activity of an immune cell.

In some embodiments, the immunotherapy is a cellular immunotherapy(e.g., adoptive T-cell therapy, dendritic cell therapy, natural killercell therapy). In some embodiments, the cellular immunotherapy issipuleucel-T (APC8015; Provenge™; Plosker (2011) Drugs 71(1): 101-108).In some embodiments, the cellular immunotherapy includes cells thatexpress a chimeric antigen receptor (CAR). In some embodiments, thecellular immunotherapy is a CAR-T cell therapy. In some embodiments, theCAR-T cell therapy is tisagenlecleucel (Kymriah™)

In some embodiments, the immunotherapy is an antibody therapy (e.g., amonoclonal antibody, a conjugated antibody). In some embodiments, theantibody therapy is bevacizumab (Mvasti™, Avastin®), trastuzumab(Herceptin®), avelumab (Bavencio®), rituximab (MabThera™, Rituxan®),edrecolomab (Panorex), daratumuab (Darzalex®), olaratumab (Lartruvo™),ofatumumab (Arzerra®), alemtuzumab (Campath®), cetuximab (Erbitux®),oregovomab, pembrolizumab (Keytruda®), dinutiximab (Unituxin®),obinutuzumab (Gazyva®), tremelimumab (CP-675,206), ramucirumab(Cyramza®), ublituximab (TG-1101), panitumumab (Vectibix®), elotuzumab(Empliciti™), avelumab (Bavencio®), necitumumab (Portrazza™),cirmtuzumab (UC-961), ibritumomab (Zevalin®), isatuximab (SAR650984),nimotuzumab, fresolimumab (GC1008), lirilumab (INN), mogamulizumab(Poteligeo®), ficlatuzumab (AV-299), denosumab (Xgeva®), ganitumab,urelumab, pidilizumab or amatuximab.

In some embodiments, the immunotherapy is an antibody-drug conjugate. Insome embodiments, the antibody-drug conjugate is gemtuzumab ozogamicin(Mylotarg™), inotuzumab ozogamicin (Besponsa®), brentuximab vedotin(Adcetris®), ado-trastuzumab emtansine (TDM-1; Kadcyla®), mirvetuximabsoravtansine (IMGN853) or anetumab ravtansine

In some embodiments, the immunotherapy includes blinatumomab (AMG103;Blincyto®) or midostaurin (Rydapt).

In some embodiments, the immunotherapy includes a toxin. In someembodiments, the immunotherapy is denileukin diftitox (Ontak®).

In some embodiments, the immunotherapy is a cytokine therapy. In someembodiments, the cytokine therapy is an interleukin 2 (IL-2) therapy, aninterferon alpha (IFNα) therapy, a granulocyte colony stimulating factor(G-CSF) therapy, an interleukin 12 (IL-12) therapy, an interleukin 15(IL-15) therapy, an interleukin 7 (IL-7) therapy or anerythropoietin-alpha (EPO) therapy. In some embodiments, the IL-2therapy is aldesleukin (Proleukin®). In some embodiments, the IFNαtherapy is IntronA® (Roferon-A®). In some embodiments, the G-CSF therapyis filgrastim (Neupogen®).

In some embodiments, the immunotherapy is an immune checkpointinhibitor. In some embodiments, the immunotherapy includes one or moreimmune checkpoint inhibitors. In some embodiments, the immune checkpointinhibitor is a CTLA-4 inhibitor, a PD-1 inhibitor or a PD-L1 inhibitor.In some embodiments, the CTLA-4 inhibitor is ipilimumab (Yervoy®) ortremelimumab (CP-675,206). In some embodiments, the PD-1 inhibitor ispembrolizumab (Keytruda®) or nivolumab (Opdivo®). In some embodiments,the PD-L1 inhibitor is atezolizumab (Tecentriq®), avelumab (Bavencio®)or durvalumab (Imfinzi™)

In some embodiments, the immunotherapy is mRNA-based immunotherapy. Insome embodiments, the mRNA-based immunotherapy is CV9104 (see, e.g.,Rausch et al. (2014) Human Vaccin Immunother 10(11): 3146-52; and Kubleret al. (2015) J. Immunother Cancer 3:26).

In some embodiments, the immunotherapy is bacillus Calmette-Guerin (BCG)therapy.

In some embodiments, the immunotherapy is an oncolytic virus therapy. Insome embodiments, the oncolytic virus therapy is talimogenealherparepvec (T-VEC; Imlygic®).

In some embodiments, the immunotherapy is a cancer vaccine. In someembodiments, the cancer vaccine is a human papillomavirus (HPV) vaccine.In some embodiments, the HPV vaccine is Gardasil®, Gardasil9® orCervarix®. In some embodiments, the cancer vaccine is a hepatitis Bvirus (HBV) vaccine. In some embodiments, the HBV vaccine is Engerix-B®,Recombivax HB® or GI-13020 (Tarmogen®). In some embodiments, the cancervaccine is Twinrix® or Pediarix®. In some embodiments, the cancervaccine is BiovaxID®, Oncophage®, GVAX, ADXS11-001, ALVAC-CEA,PROSTVAC®, Rindopepimut®, CimaVax-EGF, lapuleucel-T (APC8024;Neuvenge™), GRNVAC1, GRNVAC2, GRN-1201, hepcortespenlisimut-L(Hepko-V5), DCVAX®, SCIB 1, BMT CTN 1401, PrCa VBIR, PANVAC, ProstAtak®,DPX-Survivac, or viagenpumatucel-L (HS-110).

In some embodiments, the immunotherapy is a peptide vaccine. In someembodiments, the peptide vaccine is nelipepimut-S (E75) (NeuVax™),IMA901, or SurVaxM (SVN53-67). In some embodiments, the cancer vaccineis an immunogenic personal neoantigen vaccine (see, e.g., Ott et al.(2017) Nature 547: 217-221; Sahin et al. (2017) Nature 547: 222-226). Insome embodiments, the cancer vaccine is RGSH4K, or NEO-PV-01. In someembodiments, the cancer vaccine is a DNA-based vaccine. In someembodiments, the DNA-based vaccine is a mammaglobin-A DNA vaccine (see,e.g., Kim et al. (2016) Oncolmmunology 5(2): e1069940).

In some embodiments, immune-targeted agents are selected fromaldesleukin, interferon alfa-2b, ipilimumab, lambrolizumab, nivolumab,prednisone, and sipuleucel-T.

Non-limiting examples of radiotherapy include radioiodide therapy,external-beam radiation, and radium 223 therapy.

Additional kinase inhibitors include those described in, for example,U.S. Pat. Nos. 7,514,446; 7,863,289; 8,026,247; 8,501,756; 8,552,002;8,815,901; 8,912,204; 9,260,437; 9,273,051; U.S. Publication No. US2015/0018336; International Publication No. WO 2007/002325; WO2007/002433; WO 2008/080001; WO 2008/079906; WO 2008/079903; WO2008/079909; WO 2008/080015; WO 2009/007748; WO 2009/012283; WO2009/143018; WO 2009/143024; WO 2009/014637; 2009/152083; WO2010/111527; WO 2012/109075; WO 2014/194127; WO 2015/112806; WO2007/110344; WO 2009/071480; WO 2009/118411; WO 2010/031816; WO2010/145998; WO 2011/092120; WO 2012/101032; WO 2012/139930; WO2012/143248; WO 2012/152763; WO 2013/014039; WO 2013/102059; WO2013/050448; WO 2013/050446; WO 2014/019908; WO 2014/072220; WO2014/184069; and WO 2016/075224 all of which are hereby incorporated byreference in their entireties.

Further examples of kinase inhibitors include those described in, forexample, WO 2016/081450; WO 2016/022569; WO 2016/011141; WO 2016/011144;WO 2016/011147; WO 2015/191667; WO 2012/101029; WO 2012/113774; WO2015/191666; WO 2015/161277; WO 2015/161274; WO 2015/108992; WO2015/061572; WO 2015/058129; WO 2015/057873; WO 2015/017528;WO/2015/017533; WO 2014/160521; and WO 2014/011900, each of which ishereby incorporated by reference in its entirety.

Further examples of kinase inhibitors include luminespib (AUY-922,NVP-AUY922)(5-(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4-(4-(morpholinomethyl)phenyl)isoxazole-3-carboxamide)and doramapimod (BIRB-796)(1-[5-tert-butyl-2-(4-methylphenyl)pyrazol-3-yl]-3-[4-(2-morpholin-4-ylethoxy)naphthalen-1-yl]urea).

Accordingly, also provided herein is a method of treating cancer,comprising administering to a patient in need thereof a pharmaceuticalcombination for treating cancer which comprises (a) a compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof, (b) an additional therapeutic agent, and (c)optionally at least one pharmaceutically acceptable carrier forsimultaneous, separate or sequential use for the treatment of cancer,wherein the amounts of the compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofand the additional therapeutic agent are together effective in treatingthe cancer.

In some embodiments, the additional therapeutic agent(s) includes anyone of the above listed therapies or therapeutic agents which arestandards of care in cancers wherein the cancer has a dysregulation of aRET gene, a RET protein, or expression or activity, or level of any ofthe same.

These additional therapeutic agents may be administered with one or moredoses of the compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof, or pharmaceuticalcomposition thereof, as part of the same or separate dosage forms, viathe same or different routes of administration, and/or on the same ordifferent administration schedules according to standard pharmaceuticalpractice known to one skilled in the art.

Also provided herein is (i) a pharmaceutical combination for treating acancer in a patient in need thereof, which comprises (a) a compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof, (b) at least one additional therapeutic agent(e.g., any of the exemplary additional therapeutic agents describedherein or known in the art), and (c) optionally at least onepharmaceutically acceptable carrier for simultaneous, separate orsequential use for the treatment of cancer, wherein the amounts of thecompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof and of the additional therapeuticagent are together effective in treating the cancer; (ii) apharmaceutical composition comprising such a combination; (iii) the useof such a combination for the preparation of a medicament for thetreatment of cancer; and (iv) a commercial package or product comprisingsuch a combination as a combined preparation for simultaneous, separateor sequential use; and to a method of treatment of cancer in a patientin need thereof. In one embodiment the patient is a human. In someembodiments, the cancer is a RET-associated cancer. For example, aRET-associated cancer having one or more RET inhibitor resistancemutations.

The term “pharmaceutical combination”, as used herein, refers to apharmaceutical therapy resulting from the mixing or combining of morethan one active ingredient and includes both fixed and non-fixedcombinations of the active ingredients. The term “fixed combination”means that a compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof and at least one additionaltherapeutic agent (e.g., a chemotherapeutic agent), are bothadministered to a patient simultaneously in the form of a singlecomposition or dosage. The term “non-fixed combination” means that acompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof and at least one additionaltherapeutic agent (e.g., chemotherapeutic agent) are formulated asseparate compositions or dosages such that they may be administered to apatient in need thereof simultaneously, concurrently or sequentiallywith variable intervening time limits, wherein such administrationprovides effective levels of the two or more compounds in the body ofthe patient. These also apply to cocktail therapies, e.g. theadministration of three or more active ingredients

Accordingly, also provided herein is a method of treating a cancer,comprising administering to a patient in need thereof a pharmaceuticalcombination for treating cancer which comprises (a) a compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof, (b) an additional therapeutic agent, and (c)optionally at least one pharmaceutically acceptable carrier forsimultaneous, separate or sequential use for the treatment of cancer,wherein the amounts of the compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofand the additional therapeutic agent are together effective in treatingthe cancer. In one embodiment, the compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,and the additional therapeutic agent are administered simultaneously asseparate dosages. In one embodiment, the compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,and the additional therapeutic agent are administered as separatedosages sequentially in any order, in jointly therapeutically effectiveamounts, e.g. in daily or intermittently dosages. In one embodiment, thecompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, and the additional therapeuticagent are administered simultaneously as a combined dosage. In someembodiments, the cancer is a RET-associated cancer. For example, aRET-associated cancer having one or more RET inhibitor resistancemutations. In some embodiments, the additional therapeutic agent iscrizotinib. In some embodiments, the additional therapeutic agent isosimertinib. In some embodiments, the patient has been administered oneor more doses of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof, prior toadministration of the pharmaceutical composition. In some embodiments,the cancer is a lung cancer (e.g., a RET-associated lung cancer).

Also provided herein is a method of treating a disease or disordermediated by RET in a patient in need of such treatment, the methodcomprising administering to the patient a therapeutically effectiveamount of a compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof or a pharmaceuticalcomposition thereof. In some embodiments, the disease or disordermediated by RET is a dysregulation of RET gene, a RET kinase, orexpression or activity or level of any of the same. For example thedysregulation of a RET gene, a RET kinase, or expression or activity orlevel of any of the same includes one or more RET inhibitor resistancemutations. A disease or disorder mediated by RET can include anydisease, disorder or condition that is directly or indirectly linked toexpression or activity of RET, including overexpression and/or abnormalactivity levels. In one embodiment, the disease is cancer (e.g., aRET-associated cancer). In one embodiment, the cancer is any of thecancers or RET-associated cancers described herein. In some embodiments,the additional therapeutic agent is crizotinib. In some embodiments, theadditional therapeutic agent is osimertinib. In some embodiments, thepatient has been administered one or more doses of a compound of FormulaI-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphform thereof, prior to administration of the pharmaceutical composition.In some embodiments, the cancer is a lung cancer (e.g., a RET-associatedlung cancer).

Although the genetic basis of tumorigenesis may vary between differentcancer types, the cellular and molecular mechanisms required formetastasis appear to be similar for all solid tumor types. During ametastatic cascade, the cancer cells lose growth inhibitory responses,undergo alterations in adhesiveness and produce enzymes that can degradeextracellular matrix components. This leads to detachment of tumor cellsfrom the original tumor, infiltration into the circulation through newlyformed vasculature, migration and extravasation of the tumor cells atfavorable distant sites where they may form colonies. A number of geneshave been identified as being promoters or suppressors of metastasis.For example, overexpression of glial cell-derived neurotrophic factor(GDNF) and its RET receptor tyrosine kinase have been correlated withcancer proliferation and metastasis. See, e.g., Zeng, Q. et al. J. Int.Med. Res. (2008) 36(4): 656-64.

Accordingly, also provided herein are methods for inhibiting,preventing, aiding in the prevention, or decreasing the symptoms ofmetastasis of a cancer in a patient in need thereof, the methodcomprising administering to the patient a therapeutically effectiveamount of a compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof or a pharmaceuticalcomposition thereof. Such methods can be used in the treatment of one ormore of the cancers described herein. See, e.g., US Publication No.2013/0029925; International Publication No. WO 2014/083567; and U.S.Pat. No. 8,568,998. See also, e.g., Hezam K et al., Rev Neurosci 2018Jan. 26; 29:93-98; Gao L, et al., Pancreas 2015 January; 44:134-143;Ding K et al., J Biol Chem 2014 Jun. 6; 289:16057-71; and Amit M et al.,Oncogene 2017 Jun. 8; 36:3232-3239. In some embodiments, the cancer is aRET-associated cancer. In some embodiments, the compound of FormulaI-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphform thereof is used in combination with an additional therapy oranother therapeutic agent, including a chemotherapeutic agent, such as akinase inhibitor. For example, a first or second RET kinase inhibitor.In some embodiments, the additional therapeutic agent is crizotinib. Insome embodiments, the additional therapeutic agent is osimertinib. Insome embodiments, the patient has been administered one or more doses ofa compound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, prior to administration of thepharmaceutical composition. In some embodiments, the cancer is a lungcancer (e.g., a RET-associated lung cancer).

The term “metastasis” is an art known term and means the formation of anadditional tumor (e.g., a solid tumor) at a site distant from a primarytumor in a subject or patient, where the additional tumor includes thesame or similar cancer cells as the primary tumor.

Also provided are methods of decreasing the risk of developing ametastasis or an additional metastasis in a patient having aRET-associated cancer that include: selecting, identifying, ordiagnosing a patient as having a RET-associated cancer, andadministering a therapeutically effective amount of a compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof to the patient selected, identified, or diagnosedas having a RET-associated cancer. Also provided are methods ofdecreasing the risk of developing a metastasis or an additionalmetastasis in a patient having a RET-associated cancer that includesadministering a therapeutically effective amount of a Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofto a patient having a RET-associated cancer. The decrease in the risk ofdeveloping a metastasis or an additional metastasis in a patient havinga RET-associated cancer can be compared to the risk of developing ametastasis or an additional metastasis in the patient prior totreatment, or as compared to a patient or a population of patientshaving a similar or the same RET-associated cancer that has received notreatment or a different treatment. In some embodiments, theRET-associated cancer is a RET-associated cancer having one or more RETinhibitor resistance mutations. In some embodiments, the additionaltherapeutic agent is crizotinib. In some embodiments, the additionaltherapeutic agent is osimertinib. In some embodiments, the patient hasbeen administered one or more doses of a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,prior to administration of the pharmaceutical composition. In someembodiments, the cancer is a lung cancer (e.g., a RET-associated lungcancer).

The phrase “risk of developing a metastasis” means the risk that asubject or patient having a primary tumor will develop an additionaltumor (e.g., a solid tumor) at a site distant from a primary tumor in asubject or patient over a set period of time, where the additional tumorincludes the same or similar cancer cells as the primary tumor. Methodsfor reducing the risk of developing a metastasis in a subject or patienthaving a cancer are described herein.

The phrase “risk of developing additional metastases” means the riskthat a subject or patient having a primary tumor and one or moreadditional tumors at sites distant from the primary tumor (where the oneor more additional tumors include the same or similar cancer cells asthe primary tumor) will develop one or more further tumors distant fromthe primary tumor, where the further tumors include the same or similarcancer cells as the primary tumor. Methods for reducing the risk ofdeveloping additional metastasis are described herein.

In some embodiments, the presence of one or more RET inhibitorresistance mutations in a tumor causes the tumor to be more resistant totreatment with a first RET inhibitor. Methods useful when a RETinhibitor resistance mutation causes the tumor to be more resistant totreatment with a first RET inhibitor are described below. For example,provided herein are methods of treating a subject having a cancer thatinclude: identifying a subject having a cancer cell that has one or moreRET inhibitor resistance mutations; and administering to the identifiedsubject a compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof. In some embodiments, thecompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof is administered in combination withthe first RET inhibitor. Also provided are methods of treating a subjectidentified as having a cancer cell that has one or more RET inhibitorresistance mutations that include administering to the subject acompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof. In some embodiments, the compoundof Formula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof is administered in combination with the first RETinhibitor. In some embodiments, the one or more RET inhibitor resistancemutations confer increased resistance to a cancer cell or tumor totreatment with the first RET inhibitor. In some embodiments, the one ormore RET inhibitor resistance mutations include one or more RETinhibitor resistance mutations listed in Tables 3 and 4. For example,the one or more RET inhibitor resistance mutations can include asubstitution at amino acid position 804, e.g., V804M, V804L, or V804E,or a substitution at amino acid position 810, e.g., G810S, G810R, G810C,G810A, G810V, and G810D.

For example, provided herein are methods for treating a RET-associatedcancer in a subject in need of such treatment, the method comprising (a)detecting a dysregulation of a RET gene, a RET kinase, or the expressionor activity or level of any of the same in a sample from the subject;and (b) administering to the subject a therapeutically effective amountof a first RET inhibitor, wherein the first RET inhibitor is selectedfrom the group consisting of alectinib, cabozantinib, lenvatinib,nintedanib, ponatinib, regorfenib, sorafenib, sunitinib, vandetanib,RXDX-105 (agerafenib), BLU-667 ((1S,4R)—N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methoxy-4-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide),BLU6864, DS-5010, GSK3179106, GSK3352589, and NMS-E668. In someembodiments, the methods further comprise (after (b)) (c) determiningwhether a cancer cell in a sample obtained from the subject has at leastone RET inhibitor resistance mutation; and (d) administering a compoundof Formula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof as a monotherapy or in conjunction with anotheranticancer agent to the subject if the subject has a cancer cell thathas at least one RET inhibitor resistance mutation; or (e) administeringadditional doses of the first RET inhibitor of step (b) to the subjectif the subject has a cancer cell that does not have a RET inhibitorresistance mutation.

In some embodiments, provided herein are methods for treating aRET-associated cancer in a subject in need of such treatment, the methodcomprising (a) detecting a dysregulation of a RET gene, a RET kinase, orthe expression or activity or level of any of the same in a sample fromthe subject; and (b) administering to the subject a therapeuticallyeffective amount of a first RET inhibitor, wherein the first RETinhibitor is selected from the group consisting of alectinib,cabozantinib, lenvatinib, nintedanib, ponatinib, regorfenib, sorafenib,sunitinib, vandetanib, RXDX-105 (agerafenib), BLU-667 ((1S,4R)—N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methoxy-4-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide),BLU6864, DS-5010, GSK3179106, GSK3352589, and NMS-E668. In someembodiments, the methods further comprise (after (b)) (c) determiningwhether a cancer cell in a sample obtained from the subject has at leastone RET inhibitor resistance mutation; and (d) administering a compoundof Formula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof as a monotherapy or in conjunction with anotheranticancer agent to the subject if the subject has a cancer cell thathas at least one RET inhibitor resistance mutation; or (e) administeringadditional doses of the first RET inhibitor of step (b) to the subjectif the subject has a cancer cell that does not have a RET inhibitorresistance mutation.

In some embodiments, a compound of Formula I-IV is a polymorph form. Insome embodiments, the compound is polymorph Form A of the compound ofFormula I. In some embodiments, the compound of is polymorph Form 1 ofthe compound of Formula II. In some embodiments, the compound ispolymorph Form 2 of the compound of Formula II. In some embodiments, thecompound is polymorph Form 7 of the compound of Formula II. In someembodiments, the compound is polymorph Form 8 of the compound of FormulaII. In some embodiments, the compound is polymorph Form A of thecompound of Formula III. In some embodiments, the compound is polymorphForm A of the compound of Formula IV. In some embodiments, the compoundis polymorph Form B of the compound of Formula IV.

In some embodiments, the compound of Formula I-IV is a pharmaceuticallyacceptable salt. In some embodiments, the compound is a chloride salt ofthe compound of Formula I. In some embodiments, the compound is abromide salt of the compound of Formula I. In some embodiments, thecompound is an L-malate salt of the compound of Formula I. In someembodiments, the compound is a D-malate salt of the compound of FormulaI. In some embodiments, the compound is a phosphate salt of the compoundof Formula II. In some embodiments, the phosphate salt is asesqui-phosphate salt (e.g., 1.4:1, PO₄:free base).

In some embodiments, provided herein are methods for treating aRET-associated cancer in a subject in need of such treatment, the methodcomprising (a) detecting one or more fusion proteins of Table 1 and/orone or more RET kinase protein point mutations/insertions/deletions ofTables 2 and 2a in a sample from the subject; and (b) administering tothe subject a therapeutically effective amount of a first RET inhibitor,wherein the first RET inhibitor is selected from the group consisting ofalectinib, cabozantinib, lenvatinib, nintedanib, ponatinib, regorfenib,sorafenib, sunitinib, vandetanib, RXDX-105 (agerafenib), BLU-667 ((1S,4R)—N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methoxy-4-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide),BLU6864, DS-5010, GSK3179106, GSK3352589, and NMS-E668. In someembodiments, the methods further comprise (after (b)) (c) determiningwhether a cancer cell in a sample obtained from the subject has at leastone RET inhibitor resistance mutation of Tables 3 or 4; and (d)administering a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof, as a monotherapyor in conjunction with another anticancer agent to the subject if thesubject has a cancer cell that has at least one RET inhibitor resistancemutation; or (e) administering additional doses of the first RETinhibitor of step (b) to the subject if the subject has a cancer cellthat does not have a RET inhibitor resistance mutation.

In some embodiments, a compound of Formula I-IV is a polymorph form. Insome embodiments, the compound is polymorph Form A of the compound ofFormula I. In some embodiments, the compound of is polymorph Form 1 ofthe compound of Formula II. In some embodiments, the compound ispolymorph Form 2 of the compound of Formula II. In some embodiments, thecompound is polymorph Form 7 of the compound of Formula II. In someembodiments, the compound is polymorph Form 8 of the compound of FormulaII. In some embodiments, the compound is polymorph Form A of thecompound of Formula III. In some embodiments, the compound is polymorphForm A of the compound of Formula IV. In some embodiments, the compoundis polymorph Form B of the compound of Formula IV.

In some embodiments, the compound of Formula I-IV is a pharmaceuticallyacceptable salt. In some embodiments, the compound is a chloride salt ofthe compound of Formula I. In some embodiments, the compound is abromide salt of the compound of Formula I. In some embodiments, thecompound is an L-malate salt of the compound of Formula I. In someembodiments, the compound is a D-malate salt of the compound of FormulaI. In some embodiments, the compound is a phosphate salt of the compoundof Formula II. In some embodiments, the phosphate salt is asesqui-phosphate salt (e.g., 1.4:1, PO₄:free base).

In some embodiments, provided herein are methods for treating aRET-associated cancer in a subject in need of such treatment, the methodcomprising (a) detecting the fusion protein KIF5B-RET in a sample fromthe subject; and (b) administering to the subject a therapeuticallyeffective amount of a first RET inhibitor, wherein the first RETinhibitor is selected from the group consisting of alectinib,cabozantinib, lenvatinib, nintedanib, ponatinib, regorfenib, sorafenib,sunitinib, vandetanib, RXDX-105 (agerafenib), BLU-667((1S,4R)—N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methoxy-4-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide),BLU6864, DS-5010, GSK3179106, GSK3352589, and NMS-E668. In someembodiments, the methods further comprise (after (b)) (c) determiningwhether a cancer cell in a sample obtained from the subject has the RETinhibitor resistance mutation V804M, G810S, or G810R; and (d)administering a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof selected from thegroup consisting of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof, as a monotherapyor in conjunction with another anticancer agent to the subject if thesubject has a cancer cell that has at least one RET inhibitor resistancemutation; or (e) administering additional doses of the first RETinhibitor of step (b) to the subject if the subject has a cancer cellthat does not have a RET inhibitor resistance mutation.

In some embodiments, a compound of Formula I-IV is a polymorph form. Insome embodiments, the compound is polymorph Form A of the compound ofFormula I. In some embodiments, the compound of is polymorph Form 1 ofthe compound of Formula II. In some embodiments, the compound ispolymorph Form 2 of the compound of Formula II. In some embodiments, thecompound is polymorph Form 7 of the compound of Formula II. In someembodiments, the compound is polymorph Form 8 of the compound of FormulaII. In some embodiments, the compound is polymorph Form A of thecompound of Formula III. In some embodiments, the compound is polymorphForm A of the compound of Formula IV. In some embodiments, the compoundis polymorph Form B of the compound of Formula IV.

In some embodiments, the compound of Formula I-IV is a pharmaceuticallyacceptable salt. In some embodiments, the compound is a chloride salt ofthe compound of Formula I. In some embodiments, the compound is abromide salt of the compound of Formula I. In some embodiments, thecompound is an L-malate salt of the compound of Formula I. In someembodiments, the compound is a D-malate salt of the compound of FormulaI. In some embodiments, the compound is a phosphate salt of the compoundof Formula II. In some embodiments, the phosphate salt is asesqui-phosphate salt (e.g., 1.4:1, PO₄:free base).

As another example, provided herein are methods for treating aRET-associated cancer in a subject in need of such treatment, the methodcomprising (a) detecting a dysregulation of a RET gene, a RET kinase, orthe expression or activity or level of any of the same in a sample fromthe subject; and (b) administering to the subject a therapeuticallyeffective amount of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof. In someembodiments, the methods further comprise (after (b)) (c) determiningwhether a cancer cell in a sample obtained from the subject has at leastone RET inhibitor resistance mutation; and (d) administering a secondRET inhibitor, wherein the second RET inhibitor is selected from thegroup consisting of alectinib, cabozantinib, lenvatinib, nintedanib,ponatinib, regorfenib, sorafenib, sunitinib, vandetanib, RXDX-105(agerafenib), BLU-667((1S,4R)—N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methoxy-4-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide),BLU6864, DS-5010, GSK3179106, GSK3352589, and NMS-E668, as a monotherapyor in conjunction with another anticancer agent to the subject if thesubject has a cancer cell that has at least one RET inhibitor resistancemutation; or (e) administering additional doses of the compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof of step (b) to the subject if the subject has acancer cell that does not have a RET inhibitor resistance mutation. Insome embodiments, provided herein are methods for treating aRET-associated cancer in a subject in need of such treatment, the methodcomprising (a) detecting a dysregulation of a RET gene, a RET kinase, orthe expression or activity or level of any of the same in a sample fromthe subject; and (b) administering to the subject a therapeuticallyeffective amount of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof. In someembodiments, the methods further comprise (after (b)) (c) determiningwhether a cancer cell in a sample obtained from the subject has at leastone RET inhibitor resistance mutation; and (d) administering a secondRET inhibitor, wherein the second RET inhibitor is selected from thegroup consisting of alectinib, cabozantinib, lenvatinib, nintedanib,ponatinib, regorfenib, sorafenib, sunitinib, vandetanib, RXDX-105(agerafenib), BLU-667 ((1S,4R)—N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methoxy-4-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide),BLU6864, DS-5010, GSK3179106, GSK3352589, and NMS-E668, as a monotherapyor in conjunction with another anticancer agent to the subject if thesubject has a cancer cell that has at least one RET inhibitor resistancemutation; or (e) administering additional doses of the compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof of step (b) to the subject if the subject has acancer cell that does not have a RET inhibitor resistance mutation. Insome embodiments, provided herein are methods for treating aRET-associated cancer in a subject in need of such treatment, the methodcomprising (a) detecting one or more fusion proteins of Table 1 and/orone or more RET kinase protein point mutations/insertions/deletions ofTables 2 and 2a in a sample from the subject; and (b) administering tothe subject a therapeutically effective amount of a compound of FormulaI-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphform thereof. In some embodiments, the methods further comprise (after(b)) (c) determining whether a cancer cell in a sample obtained from thesubject has at least one RET inhibitor resistance mutation of Tables 3or 4; and (d) administering a second RET inhibitor, wherein the secondRET inhibitor is selected from the group consisting of alectinib,cabozantinib, lenvatinib, nintedanib, ponatinib, regorfenib, sorafenib,sunitinib, vandetanib, RXDX-105 (agerafenib), BLU-667((1S,4R)—N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methoxy-4-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide),BLU6864, DS-5010, GSK3179106, GSK3352589, and NMS-E668, as a monotherapyor in conjunction with another anticancer agent to the subject if thesubject has a cancer cell that has at least one RET inhibitor resistancemutation; or (e) administering additional doses of the compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof of step (b) to the subject if the subject has acancer cell that does not have a RET inhibitor resistance mutation. Insome embodiments, provided herein are methods for treating aRET-associated cancer in a subject in need of such treatment, the methodcomprising (a) detecting the fusion protein KIF5B-RET in a sample fromthe subject; and (b) administering to the subject a therapeuticallyeffective amount of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof. In someembodiments, the methods further comprise (after (b)) (c) determiningwhether a cancer cell in a sample obtained from the subject has the RETinhibitor resistance mutation V804M, G810S, or G810R; and (d)administering a second RET inhibitor, wherein the second RET inhibitoris selected from the group consisting of alectinib, cabozantinib,lenvatinib, nintedanib, ponatinib, regorfenib, sorafenib, sunitinib,vandetanib, RXDX-105 (agerafenib), BLU-667 ((1S,4R)—N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methoxy-4-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide),BLU6864, DS-5010, GSK3179106, GSK3352589, and NMS-E668, as a monotherapyor in conjunction with another anticancer agent to the subject if thesubject has a cancer cell that has at least one RET inhibitor resistancemutation; or (e) administering additional doses of the compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof of step (b) to the subject if the subject has acancer cell that does not have a RET inhibitor resistance mutation.

As another example, provided herein are methods for treating aRET-associated cancer in a subject in need of such treatment, the methodcomprising (a) detecting a dysregulation of a RET gene, a RET kinase, orthe expression or activity or level of any of the same in a sample fromthe subject; and (b) administering to the subject a therapeuticallyeffective amount of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof. In someembodiments, the methods further comprise (after (b)) (c) determiningwhether a cancer cell in a sample obtained from the subject has at leastone RET inhibitor resistance mutation; and (d) administering a secondtherapeutic agent, wherein the second therapeutic agent is selected fromthe group consisting of crizotinib and osimertinib, as a monotherapy orin conjunction with a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof to the subject ifthe subject has a cancer cell that has at least one RET inhibitorresistance mutation; or (e) administering additional doses of thecompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof of step (b) to the subject if thesubject has a cancer cell that does not have a RET inhibitor resistancemutation. In some embodiments, provided herein are methods for treatinga RET-associated cancer in a subject in need of such treatment, themethod comprising (a) detecting one or more fusion proteins of Table 1and/or one or more RET kinase protein point mutations/insertions ofTable 2 in a sample from the subject; and (b) administering to thesubject a therapeutically effective amount of a compound of FormulaI-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphform thereof. In some embodiments, the methods further comprise (after(b)) (c) determining whether a cancer cell in a sample obtained from thesubject has at least one RET inhibitor resistance mutation of Tables 3or 4; and (d) administering a second therapeutic agent, wherein thesecond therapeutic agent is selected from the group consisting ofcrizotinib and osimertinib, as a monotherapy or in conjunction with acompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof to the subject if the subject has acancer cell that has at least one RET inhibitor resistance mutation; or(e) administering additional doses of the compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofof step (b) to the subject if the subject has a cancer cell that doesnot have a RET inhibitor resistance mutation. In some embodiments of theabove, the RET-associated cancer is a lung cancer.

In some embodiments, the presence of one or more RET inhibitorresistance mutations in a tumor causes the tumor to be more resistant totreatment with a first RET inhibitor. Methods useful when a RETinhibitor resistance mutation causes the tumor to be more resistant totreatment with a first RET inhibitor are described below. For example,provided herein are methods of treating a subject having a cancer thatinclude: identifying a subject having a cancer cell that has one or moreRET inhibitor resistance mutations; and administering to the identifiedsubject a compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof. In some embodiments, thecompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof is administered in combination withthe first RET inhibitor. Also provided are methods of treating a subjectidentified as having a cancer cell that has one or more RET inhibitorresistance mutations that include administering to the subject acompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof. In some embodiments, the compoundof Formula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof is administered in combination with the first RETinhibitor. In some embodiments, the one or more RET inhibitor resistancemutations confer increased resistance to a cancer cell or tumor totreatment with the first RET inhibitor. In some embodiments, the one ormore RET inhibitor resistance mutations include one or more RETinhibitor resistance mutations listed in Tables 3 and 4. For example,the one or more RET inhibitor resistance mutations can include asubstitution at amino acid position 804, e.g., V804M, V804L, or V804E,or a substitution at amino acid position 810, e.g., G810S, G810R, G810C,G810A, G810V, and G810D.

In some embodiments provided herein, circulating tumor DNA can be usedto monitor the responsiveness of a patient to a particular therapy(e.g., a first RET inhibitor, a second RET inhibitor, or a compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof). For example, prior to starting treatment with atherapy as described herein (e.g., a first RET inhibitor, a second RETinhibitor, or a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof), a biologicalsample can be obtained from the subject and the level of circulatingtumor DNA determined in the biological sample. This sample can beconsidered a base-line sample. The subject can then be administered oneor more doses of a therapy as described herein (e.g., a first RETinhibitor, a second RET inhibitor, or a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof)and the levels of circulating tumor DNA can be monitored (e.g., afterthe first dose, second dose, third dose, etc. or after one week, twoweeks, three weeks, four weeks, etc.). If the level of circulating tumorDNA is lower than the baseline sample (e.g., a 1% to about a 99%reduction, a 1% to about a 95% reduction, a 1% to about a 90% reduction,a 1% to about a 85% reduction, a 1% to about a 80% reduction, a 1% toabout a 75% reduction, a 1% reduction to about a 70% reduction, a 1%reduction to about a 65% reduction, a 1% reduction to about a 60%reduction, a 1% reduction to about a 55% reduction, a 1% reduction toabout a 50% reduction, a 1% reduction to about a 45% reduction, a 1%reduction to about a 40% reduction, a 1% reduction to about a 35%reduction, a 1% reduction to about a 30% reduction, a 1% reduction toabout a 25% reduction, a 1% reduction to about a 20% reduction, a 1%reduction to about a 15% reduction, a 1% reduction to about a 10%reduction, a 1% to about a 5% reduction, about a 5% to about a 99%reduction, about a 10% to about a 99% reduction, about a 15% to about a99% reduction, about a 20% to about a 99% reduction, about a 25% toabout a 99% reduction, about a 30% to about a 99% reduction, about a 35%to about a 99% reduction, about a 40% to about a 99% reduction, about a45% to about a 99% reduction, about a 50% to about a 99% reduction,about a 55% to about a 99% reduction, about a 60% to about a 99%reduction, about a 65% to about a 99% reduction, about a 70% to about a99% reduction, about a 75% to about a 95% reduction, about a 80% toabout a 99% reduction, about a 90% reduction to about a 99% reduction,about a 95% to about a 99% reduction, about a 5% to about a 10%reduction, about a 5% to about a 25% reduction, about a 10% to about a30% reduction, about a 20% to about a 40% reduction, about a 25% toabout a 50% reduction, about a 35% to about a 55% reduction, about a 40%to about a 60% reduction, about a 50% reduction to about a 75%reduction, about a 60% reduction to about 80% reduction, or about a 65%to about a 85% reduction etc.), this is indicative of responsiveness tothe therapy. In some embodiments, the level of circulating tumor DNA isreduced such that it is below the detection limit of the instrument. Insome embodiments, the level of circulating tumor DNA in a biologicalsample obtained from the patient (n) is compared to the sample takenjust previous (n−1). If the level of circulating tumor DNA in the nsample is lower than the n−1 sample (e.g., a 1% to about a 99%reduction, a 1% to about a 95% reduction, a 1% to about a 90% reduction,a 1% to about a 85% reduction, a 1% to about a 80% reduction, a 1% toabout a 75% reduction, a 1% reduction to about a 70% reduction, a 1%reduction to about a 65% reduction, a 1% reduction to about a 60%reduction, a 1% reduction to about a 55% reduction, a 1% reduction toabout a 50% reduction, a 1% reduction to about a 45% reduction, a 1%reduction to about a 40% reduction, a 1% reduction to about a 35%reduction, a 1% reduction to about a 30% reduction, a 1% reduction toabout a 25% reduction, a 1% reduction to about a 20% reduction, a 1%reduction to about a 15% reduction, a 1% reduction to about a 10%reduction, a 1% to about a 5% reduction, about a 5% to about a 99%reduction, about a 10% to about a 99% reduction, about a 15% to about a99% reduction, about a 20% to about a 99% reduction, about a 25% toabout a 99% reduction, about a 30% to about a 99% reduction, about a 35%to about a 99% reduction, about a 40% to about a 99% reduction, about a45% to about a 99% reduction, about a 50% to about a 99% reduction,about a 55% to about a 99% reduction, about a 60% to about a 99%reduction, about a 65% to about a 99% reduction, about a 70% to about a99% reduction, about a 75% to about a 95% reduction, about a 80% toabout a 99% reduction, about a 90% reduction to about a 99% reduction,about a 95% to about a 99% reduction, about a 5% to about a 10%reduction, about a 5% to about a 25% reduction, about a 10% to about a30% reduction, about a 20% to about a 40% reduction, about a 25% toabout a 50% reduction, about a 35% to about a 55% reduction, about a 40%to about a 60% reduction, about a 50% reduction to about a 75%reduction, about a 60% reduction to about 80% reduction, or about a 65%to about a 85% reduction, etc.), this is indicative of responsiveness tothe therapy. In some embodiments, the level of circulating tumor DNA isreduced such that it is below the detection limit of the instrument. Inthe case of responsiveness to therapy, the subject can to beadministered one or more doses of the therapy and the circulating tumorDNA can be continued to be monitored.

If the level of circulating tumor DNA in the sample is higher than thebaseline (e.g., a 1% to about a 99% increase, a 1% to about a 95%increase, a 1% to about a 90% increase, a 1% to about a 85% increase, a1% to about a 80% increase, a 1% to about a 75% increase, a 1% increaseto about a 70% increase, a 1% increase to about a 65% increase, a 1%increase to about a 60% increase, a 1% increase to about a 55% increase,a 1% increase to about a 50% increase, a 1% increase to about a 45%increase, a 1% increase to about a 40% increase, a 1% increase to abouta 35% increase, a 1% increase to about a 30% increase, a 1% increase toabout a 25% increase, a 1% increase to about a 20% increase, a 1%increase to about a 15% increase, a 1% increase to about a 10% increase,a 1% to about a 5% increase, about a 5% to about a 99% increase, about a10% to about a 99% increase, about a 15% to about a 99% increase, abouta 20% to about a 99% increase, about a 25% to about a 99% increase,about a 30% to about a 99% increase, about a 35% to about a 99%increase, about a 40% to about a 99% increase, about a 45% to about a99% increase, about a 50% to about a 99% increase, about a 55% to abouta 99% increase, about a 60% to about a 99% increase, about a 65% toabout a 99% increase, about a 70% to about a 99% increase, about a 75%to about a 95% increase, about a 80% to about a 99% increase, about a90% increase to about a 99% increase, about a 95% to about a 99%increase, about a 5% to about a 10% increase, about a 5% to about a 25%increase, about a 10% to about a 30% increase, about a 20% to about a40% increase, about a 25% to about a 50% increase, about a 35% to abouta 55% increase, about a 40% to about a 60% increase, about a 50%increase to about a 75% increase, about a 60% increase to about 80%increase, or about a 65% to about a 85% increase, etc.), this can beindicative of resistance to the therapy. If the level of circulatingtumor DNA in the n sample is higher than the n−1 sample (e.g., a 1% toabout a 99% increase, a 1% to about a 95% increase, a 1% to about a 90%increase, a 1% to about a 85% increase, a 1% to about a 80% increase, a1% to about a 75% increase, a 1% increase to about a 70% increase, a 1%increase to about a 65% increase, a 1% increase to about a 60% increase,a 1% increase to about a 55% increase, a 1% increase to about a 50%increase, a 1% increase to about a 45% increase, a 1% increase to abouta 40% increase, a 1% increase to about a 35% increase, a 1% increase toabout a 30% increase, a 1% increase to about a 25% increase, a 1%increase to about a 20% increase, a 1% increase to about a 15% increase,a 1% increase to about a 10% increase, a 1% to about a 5% increase,about a 5% to about a 99% increase, about a 10% to about a 99% increase,about a 15% to about a 99% increase, about a 20% to about a 99%increase, about a 25% to about a 99% increase, about a 30% to about a99% increase, about a 35% to about a 99% increase, about a 40% to abouta 99% increase, about a 45% to about a 99% increase, about a 50% toabout a 99% increase, about a 55% to about a 99% increase, about a 60%to about a 99% increase, about a 65% to about a 99% increase, about a70% to about a 99% increase, about a 75% to about a 95% increase, abouta 80% to about a 99% increase, about a 90% increase to about a 99%increase, about a 95% to about a 99% increase, about a 5% to about a 10%increase, about a 5% to about a 25% increase, about a 10% to about a 30%increase, about a 20% to about a 40% increase, about a 25% to about a50% increase, about a 35% to about a 55% increase, about a 40% to abouta 60% increase, about a 50% increase to about a 75% increase, about a60% increase to about 80% increase, or about a 65% to about a 85%increase etc.), this can be indicative of resistance to the therapy.When resistance to therapy is suspected, the subject can undergo one ormore of imaging, biopsy, surgery, or other diagnostic tests. In someembodiments, when resistance to the therapy is suspected, the subjectcan be administered (either as a monotherapy or in combination with theprevious therapy) a compound capable of treating a RET inhibitorresistance (e.g., a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof, as providedherein). See, for example, Cancer Discov; 7(12); 1368-70 (2017); andCancer Discov; 7(12); 1394-403 (2017).

In some embodiments provided herein, a protein biomarker can be used tomonitor the responsiveness of a patient to a particular therapy (e.g., afirst RET inhibitor, a second RET inhibitor, or a compound of FormulaI-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphform thereof). For example, prior to starting treatment with a therapyas described herein (e.g., a first RET inhibitor, a second RETinhibitor, or a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof), a biologicalsample can be obtained from the subject and the level of a proteinbiomarker can be determined in the biological sample. This sample can beconsidered a base-line sample. The subject can then be administered oneor more doses of a therapy as described herein (e.g., a first RETinhibitor, a second RET inhibitor, or a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof)and the levels of the protein biomarker can be monitored (e.g., afterthe first dose, second dose, third dose, etc. or after one week, twoweeks, three weeks, four weeks, etc.). If the level of the proteinbiomarker is lower than the baseline sample (e.g., a 1% to about a 99%reduction, a 1% to about a 95% reduction, a 1% to about a 90% reduction,a 1% to about a 85% reduction, a 1% to about a 80% reduction, a 1% toabout a 75% reduction, a 1% reduction to about a 70% reduction, a 1%reduction to about a 65% reduction, a 1% reduction to about a 60%reduction, a 1% reduction to about a 55% reduction, a 1% reduction toabout a 50% reduction, a 1% reduction to about a 45% reduction, a 1%reduction to about a 40% reduction, a 1% reduction to about a 35%reduction, a 1% reduction to about a 30% reduction, a 1% reduction toabout a 25% reduction, a 1% reduction to about a 20% reduction, a 1%reduction to about a 15% reduction, a 1% reduction to about a 10%reduction, a 1% to about a 5% reduction, about a 5% to about a 99%reduction, about a 10% to about a 99% reduction, about a 15% to about a99% reduction, about a 20% to about a 99% reduction, about a 25% toabout a 99% reduction, about a 30% to about a 99% reduction, about a 35%to about a 99% reduction, about a 40% to about a 99% reduction, about a45% to about a 99% reduction, about a 50% to about a 99% reduction,about a 55% to about a 99% reduction, about a 60% to about a 99%reduction, about a 65% to about a 99% reduction, about a 70% to about a99% reduction, about a 75% to about a 95% reduction, about a 80% toabout a 99% reduction, about a 90% reduction to about a 99% reduction,about a 95% to about a 99% reduction, about a 5% to about a 10%reduction, about a 5% to about a 25% reduction, about a 10% to about a30% reduction, about a 20% to about a 40% reduction, about a 25% toabout a 50% reduction, about a 35% to about a 55% reduction, about a 40%to about a 60% reduction, about a 50% reduction to about a 75%reduction, about a 60% reduction to about 80% reduction, or about a 65%to about a 85% reduction etc.), this is indicative of responsiveness tothe therapy. In some embodiments, the level of the protein biomarker isreduced such that it is below the detection limit of the instrument. Insome embodiments, the level of the protein biomarker in a biologicalsample obtained from the patient (n) is compared to the sample takenjust previous (n−1). If the level of the protein biomarker in the nsample is lower than the n−1 sample (e.g., a 1% to about a 99%reduction, a 1% to about a 95% reduction, a 1% to about a 90% reduction,a 1% to about a 85% reduction, a 1% to about a 80% reduction, a 1% toabout a 75% reduction, a 1% reduction to about a 70% reduction, a 1%reduction to about a 65% reduction, a 1% reduction to about a 60%reduction, a 1% reduction to about a 55% reduction, a 1% reduction toabout a 50% reduction, a 1% reduction to about a 45% reduction, a 1%reduction to about a 40% reduction, a 1% reduction to about a 35%reduction, a 1% reduction to about a 30% reduction, a 1% reduction toabout a 25% reduction, a 1% reduction to about a 20% reduction, a 1%reduction to about a 15% reduction, a 1% reduction to about a 10%reduction, a 1% to about a 5% reduction, about a 5% to about a 99%reduction, about a 10% to about a 99% reduction, about a 15% to about a99% reduction, about a 20% to about a 99% reduction, about a 25% toabout a 99% reduction, about a 30% to about a 99% reduction, about a 35%to about a 99% reduction, about a 40% to about a 99% reduction, about a45% to about a 99% reduction, about a 50% to about a 99% reduction,about a 55% to about a 99% reduction, about a 60% to about a 99%reduction, about a 65% to about a 99% reduction, about a 70% to about a99% reduction, about a 75% to about a 95% reduction, about a 80% toabout a 99% reduction, about a 90% reduction to about a 99% reduction,about a 95% to about a 99% reduction, about a 5% to about a 10%reduction, about a 5% to about a 25% reduction, about a 10% to about a30% reduction, about a 20% to about a 40% reduction, about a 25% toabout a 50% reduction, about a 35% to about a 55% reduction, about a 40%to about a 60% reduction, about a 50% reduction to about a 75%reduction, about a 60% reduction to about 80% reduction, or about a 65%to about a 85% reduction, etc.), this is indicative of responsiveness tothe therapy. In some embodiments, the level of the protein biomarker isreduced such that it is below the detection limit of the instrument. Inthe case of responsiveness to therapy, the subject can to beadministered one or more doses of the therapy and the protein biomarkercan be continued to be monitored.

If the level of the protein biomarker in the sample is higher than thebaseline (e.g., a 1% to about a 99% increase, a 1% to about a 95%increase, a 1% to about a 90% increase, a 1% to about a 85% increase, a1% to about a 80% increase, a 1% to about a 75% increase, a 1% increaseto about a 70% increase, a 1% increase to about a 65% increase, a 1%increase to about a 60% increase, a 1% increase to about a 55% increase,a 1% increase to about a 50% increase, a 1% increase to about a 45%increase, a 1% increase to about a 40% increase, a 1% increase to abouta 35% increase, a 1% increase to about a 30% increase, a 1% increase toabout a 25% increase, a 1% increase to about a 20% increase, a 1%increase to about a 15% increase, a 1% increase to about a 10% increase,a 1% to about a 5% increase, about a 5% to about a 99% increase, about a10% to about a 99% increase, about a 15% to about a 99% increase, abouta 20% to about a 99% increase, about a 25% to about a 99% increase,about a 30% to about a 99% increase, about a 35% to about a 99%increase, about a 40% to about a 99% increase, about a 45% to about a99% increase, about a 50% to about a 99% increase, about a 55% to abouta 99% increase, about a 60% to about a 99% increase, about a 65% toabout a 99% increase, about a 70% to about a 99% increase, about a 75%to about a 95% increase, about a 80% to about a 99% increase, about a90% increase to about a 99% increase, about a 95% to about a 99%increase, about a 5% to about a 10% increase, about a 5% to about a 25%increase, about a 10% to about a 30% increase, about a 20% to about a40% increase, about a 25% to about a 50% increase, about a 35% to abouta 55% increase, about a 40% to about a 60% increase, about a 50%increase to about a 75% increase, about a 60% increase to about 80%increase, or about a 65% to about a 85% increase, etc.), this can beindicative of resistance to the therapy. If the level of the proteinbiomarker in the n sample is higher than the n−1 sample (e.g., a 1% toabout a 99% increase, a 1% to about a 95% increase, a 1% to about a 90%increase, a 1% to about a 85% increase, a 1% to about a 80% increase, a1% to about a 75% increase, a 1% increase to about a 70% increase, a 1%increase to about a 65% increase, a 1% increase to about a 60% increase,a 1% increase to about a 55% increase, a 1% increase to about a 50%increase, a 1% increase to about a 45% increase, a 1% increase to abouta 40% increase, a 1% increase to about a 35% increase, a 1% increase toabout a 30% increase, a 1% increase to about a 25% increase, a 1%increase to about a 20% increase, a 1% increase to about a 15% increase,a 1% increase to about a 10% increase, a 1% to about a 5% increase,about a 5% to about a 99% increase, about a 10% to about a 99% increase,about a 15% to about a 99% increase, about a 20% to about a 99%increase, about a 25% to about a 99% increase, about a 30% to about a99% increase, about a 35% to about a 99% increase, about a 40% to abouta 99% increase, about a 45% to about a 99% increase, about a 50% toabout a 99% increase, about a 55% to about a 99% increase, about a 60%to about a 99% increase, about a 65% to about a 99% increase, about a70% to about a 99% increase, about a 75% to about a 95% increase, abouta 80% to about a 99% increase, about a 90% increase to about a 99%increase, about a 95% to about a 99% increase, about a 5% to about a 10%increase, about a 5% to about a 25% increase, about a 10% to about a 30%increase, about a 20% to about a 40% increase, about a 25% to about a50% increase, about a 35% to about a 55% increase, about a 40% to abouta 60% increase, about a 50% increase to about a 75% increase, about a60% increase to about 80% increase, or about a 65% to about a 85%increase etc.), this can be indicative of resistance to the therapy.When resistance to therapy is suspected, the subject can undergo one ormore of imaging, biopsy, surgery, or other diagnostic tests. In someembodiments, when resistance to the therapy is suspected, the subjectcan be administered (either as a monotherapy or in combination with theprevious therapy) a compound capable of treating a RET inhibitorresistance (e.g., a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof, as providedherein).

In some embodiments, one or more protein biomarkers are monitored. Theparticular protein biomarkers to be monitored can depend on the type ofcancer and can be readily identified by one having ordinary skill in theart. Non-limiting examples of protein biomarkers include: CA 125,carcinoembryonic antigen (CEA), calcitonin, thyroglobulin,adrenocorticotropic hormone (ACTH), cortisol, CA 19-9, prolactin,hepatocyte growth factor, osteopontin, myeloperoxidase, tissue inhibitorof metalloproteinases 1, angiopoietin-1 (Ang-1), cytokeratin 19 (CK-19),tissue inhibitor of metalloproteinase-1 (TIMP-1), chitinase 3 like-1(YKL-40), galectin-3 (GAL-3), CYFRA 21-1 (cytokeratins), EPCAM(epithelial cell adhesion molecule), ProGRP (pro-gastrin-releasingpeptide), and CEACAM (carcinoembryonic antigen). See, for example, CohenJ D, Li L, Wang Y, et al. Detection and localization of surgicallyresectable cancers with a multi-analyte blood test. Science; Publishedonline 18 Jan. 2018. pii: eaar3247. DOI: 10.1126/science.aar3247; FawazM Makki et al. Serum biomarkers of papillary thyroid cancer. JOtolaryngol Head Neck Surg. 2013; 42(1): 16; and Tatiana N. Zamay et al.Current and Prospective Protein Biomarkers of Lung Cancer. Cancers(Basel). 2017 November; 9(11): 155. In some embodiments, the biomarkersinclude one or more of CEA, calcitonin, thyroglobulin, ACTH, andcortisol. In some embodiments, the cancer is medullary thyroid cancerand the protein biomarkers include CEA and calcitonin. In someembodiments, the cancer is non-medullary thyroid cancer and the proteinbiomarker include thyroglobulin. In some embodiments, the biomarkers areACTH and cortisol (e.g., when a patient as Cushing's disease related totheir cancer).

Also provided herein are methods of treating a RET-associated cancer ina subject that include (a) administering one or more (e.g., two or more,three or more, four or more, five or more, or ten or more) doses of afirst RET kinase inhibitor to a subject identified or diagnosed ashaving a RET-associated cancer (e.g., any of the types of RET-associatedcancers described herein)(e.g., identified or diagnosed as having aRET-associated cancer using any of the exemplary methods describedherein or known in the art); (b) after step (a), determining a level ofcirculating tumor DNA in a biological sample (e.g., a biological samplecomprising blood, serum, or plasma) obtained from the subject; (c)administering a therapeutically effective amount of a second RETinhibitor or a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof as a monotherapyor in conjunction with another anticancer agent to a subject identifiedas having about the same or an elevated level of circulating tumor DNAas compared to a reference level of circulating tumor DNA (e.g., any ofthe reference levels of circulating tumor DNA described herein). In someexamples of these methods, the reference level of circulating tumor DNAis a level of circulating tumor DNA in a biological sample obtained fromthe subject prior to step (a). Some embodiments of these methods furtherinclude determining the level of circulating tumor DNA in the biologicalsample obtained from the subject prior to step (a). In some examples ofthese methods, the reference level of circulating tumor DNA is athreshold level of circulating tumor DNA (e.g., an average level ofcirculating tumor DNA in a population of subjects having a similarRET-associated cancer and having a similar stage of the RET-associatedcancer, but receiving a non-effective treatment or a placebo, or not yetreceiving therapeutic treatment, or a level of circulating tumor DNA ina subject having a similar RET-associated cancer and having a similarstage of the RET-associated cancer, but receiving a non-effectivetreatment or a placebo, or not yet receiving therapeutic treatment). Insome examples of these methods, the first RET inhibitor is selected fromthe group of: cabozantinib, vandetanib, alectinib, apatinib,sitravatinib, sorafenib, lenvatinib, ponatinib, dovitinib, sunitinib,foretinib, BLU667, and BLU6864.

Also provided herein are methods of treating a RET-associated cancer ina subject that include administering a therapeutically effective amountof a compound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, to a subject (i) identified ordiagnosed as having a RET-associated cancer (e.g., any of the types ofRET-associated cancers described herein) (e.g., identified or diagnosedas having a RET-associated cancer using any of the exemplary methodsdescribed herein or known in the art), (ii) previously administered oneor more (e.g., two or more, three or more, four or more, five or more,or ten or more) doses of a second RET kinase inhibitor, and (ii) afterthe prior administration of the one or more doses of the second RETkinase inhibitor, identified as having about the same or an elevatedlevel of circulating tumor DNA as compared to a reference level ofcirculating tumor DNA (e.g., any of the reference levels of circulatingtumor DNA described herein or known in the art). In some embodiments ofthese methods, the reference level of circulating tumor DNA is a levelof circulating tumor DNA in a biological sample (e.g., a biologicalsample comprising blood, plasma, or serum) obtained from the subjectprior to the administration of the one or more doses of the second RETkinase inhibitor. Some embodiments of these methods further includedetermining the level of circulating tumor DNA in the biological sampleobtained from the subject prior to administration of the one or moredoses of the second RET kinase inhibitor. In some examples of thesemethods, the reference level of circulating tumor DNA is a thresholdlevel of circulating tumor DNA (e.g., an average level of circulatingtumor DNA in a population of subjects having a similar RET-associatedcancer and having a similar stage of the RET-associated cancer, butreceiving a non-effective treatment or a placebo, or not yet receivingtherapeutic treatment, or a level of circulating tumor DNA in a subjecthaving a similar RET-associated cancer and having a similar stage of theRET-associated cancer, but receiving a non-effective treatment or aplacebo, or not yet receiving therapeutic treatment). In someembodiments of these methods, the second RET kinase inhibitor isselected from the group consisting of: cabozantinib, vandetanib,alectinib, apatinib, sitravatinib, sorafenib, lenvatinib, ponatinib,dovitinib, sunitinib, foretinib, BLU667, and BLU6864.

Also provided herein are methods of treating a RET-associated cancer ina subject that include: (a) administering one or more doses of acompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, as a monotherapy to a subjectidentified or diagnosed as having a RET-associated cancer (e.g., any ofthe types of RET-associated cancer described herein) (e.g., a subjectidentified or diagnosed as having a RET-associated cancer using any ofthe methods described herein or known in the art); (b) after step (a),determining a level of circulating tumor DNA in a biological sample(e.g., a biological sample comprising blood, serum, or plasma) obtainedfrom the subject; (c) administering a therapeutically effective amountof a compound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, and an additional therapeuticagent or treatment (e.g., any of the additional therapeutic agents ortreatments of a RET-associated cancer described herein or known in theart) to a subject identified as having about the same or an elevatedlevel of circulating tumor DNA as compared to a reference level ofcirculating tumor DNA (e.g., any of the exemplary reference levels ofcirculating tumor DNA described herein or known in the art). In someembodiments of these methods, the additional therapeutic agent is asecond RET kinase inhibitor (e.g., a RET kinase inhibitor selected fromthe group of: cabozantinib, vandetanib, alectinib, apatinib,sitravatinib, sorafenib, lenvatinib, ponatinib, dovitinib, sunitinib,foretinib, BLU667, and BLU6864. In some examples of any of thesemethods, the additional therapeutic agent or treatment comprises one ormore of: radiation therapy, a chemotherapeutic agent (e.g., any of theexemplary chemotherapeutic agents described herein or known in the art),a checkpoint inhibitor (e.g., any of the exemplary checkpoint inhibitorsdescribed herein or known in the art), surgery (e.g., at least partialresection of the tumor) and one or more other kinase inhibitors (e.g.,any of the exemplary kinase inhibitors described herein or known in theart). In some examples of these methods, the reference level ofcirculating tumor DNA is a level of circulating tumor DNA in abiological sample (e.g., a biological sample comprising blood, serum, orplasma) obtained from the subject prior to step (a). In some examples ofthese methods, the reference level of circulating tumor DNA is athreshold level of circulating tumor DNA (e.g., an average level ofcirculating tumor DNA in a population of subjects having a similarRET-associated cancer and having a similar stage of the RET-associatedcancer, but receiving a non-effective treatment or a placebo, or not yetreceiving therapeutic treatment, or a level of circulating tumor DNA ina subject having a similar RET-associated cancer and having a similarstage of the RET-associated cancer, but receiving a non-effectivetreatment or a placebo, or not yet receiving therapeutic treatment).

Also provided herein are methods of treating a RET-associated cancer ina subject that include: administering a therapeutically effective amountof a compound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, and an additional therapeuticagent or treatment to a subject (i) identified or diagnosed as having aRET-associated cancer (e.g., any of the types of RET-associated cancerdescribed herein) (e.g., a subject identified or diagnosed as having aRET-associated cancer using any of the methods described herein or knownin the art), (ii) previously administered one or more doses of thecompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, as a monotherapy, and (ii) afteradministration of the one or more (e.g., two or more, three or more,four or more, five or more, or ten or more) doses of the compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof, as a monotherapy, identified as having about thesame or an elevated level of circulating tumor DNA as compared to areference level of circulating tumor DNA (e.g., any of the exemplaryreference levels of circulating tumor DNA described herein). In someembodiments of these methods, the reference level of circulating tumorDNA is a level of circulating tumor DNA in a biological sample obtainedfrom the subject prior to administration of the one or more (e.g., twoor more, three or more, four or more, five or more, or ten or more)doses of the compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof, as a monotherapy. Someembodiments of these methods further include determining the level ofcirculating tumor DNA in the biological sample obtained from the subjectprior to administration of the one or more doses of the compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof, as a monotherapy. In some examples of thesemethods, the reference level of circulating tumor DNA is a thresholdlevel of circulating tumor DNA (e.g., an average level of circulatingtumor DNA in a population of subjects having a similar RET-associatedcancer and having a similar stage of the RET-associated cancer, butreceiving a non-effective treatment or a placebo, or not yet receivingtherapeutic treatment, or a level of circulating tumor DNA in a subjecthaving a similar RET-associated cancer and having a similar stage of theRET-associated cancer, but receiving a non-effective treatment or aplacebo, or not yet receiving therapeutic treatment). In someembodiments of this method, the additional therapeutic agent is a secondRET kinase inhibitor (e.g., a second RET kinase inhibitor selected fromthe group of cabozantinib, vandetanib, alectinib, apatinib,sitravatinib, sorafenib, lenvatinib, ponatinib, dovitinib, sunitinib,foretinib, BLU667, and BLU6864. In some embodiments of these methods,the additional therapeutic agent or treatment includes one or more ofradiation therapy, a chemotherapeutic agent (e.g., any of the exemplarychemotherapeutic agents described herein or known in the art), acheckpoint inhibitor (e.g., any of the exemplary checkpoint inhibitorsdescribed herein or known in the art), surgery (e.g., at least partialresection of the tumor), and one or more other kinase inhibitors (e.g.,any of the kinase inhibitors described herein or known in the art).

Also provided herein are methods of selecting a treatment for a subjectthat include: selecting a therapeutically effective amount of a compoundof Formula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof, for a subject (i) identified or diagnosed ashaving a RET-associated cancer (e.g., any of the RET-associated cancersdescribed herein) (e.g., a subject identified or diagnosed as having aRET-associated cancer using any of the methods described herein or knownin the art), (ii) previously administered one or more (e.g., two ormore, three or more, four or more, five or more, or ten or more) dosesof a second RET kinase inhibitor (e.g., any of the RET kinase inhibitorsdescribed herein or known in the art), and (ii) after administration ofthe one or more doses of the second RET kinase inhibitor, identified ashaving about the same or an elevated level of circulating tumor DNA ascompared to a reference level of circulating tumor DNA. In someembodiments of any of these methods, the reference level of circulatingtumor DNA is a level of circulating tumor DNA in a biological sample(e.g., a biological sample comprising blood, serum, or plasma) obtainedfrom the subject prior to administration of the one or more doses of thesecond RET kinase inhibitor. Some embodiments of these methods furtherinclude determining the level of circulating tumor DNA in the biologicalsample obtained from the subject prior to administration of the one ormore doses of the second RET kinase inhibitor. In some examples of thesemethods, the reference level of circulating tumor DNA is a thresholdlevel of circulating tumor DNA (e.g., an average level of circulatingtumor DNA in a population of subjects having a similar RET-associatedcancer and having a similar stage of the RET-associated cancer, butreceiving a non-effective treatment or a placebo, or not yet receivingtherapeutic treatment, or a level of circulating tumor DNA in a subjecthaving a similar RET-associated cancer and having a similar stage of theRET-associated cancer, but receiving a non-effective treatment or aplacebo, or not yet receiving therapeutic treatment). In someembodiments of any these methods, the second RET kinase inhibitor isselected from the group of cabozantinib, vandetanib, alectinib,apatinib, sitravatinib, sorafenib, lenvatinib, ponatinib, dovitinib,sunitinib, foretinib, BLU667, and BLU6864.

Also provided herein are methods of selecting a treatment for a subjectthat include selecting a therapeutically effective amount of a compoundof Formula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof, and an additional therapeutic agent or treatmentfor a subject (i) identified or diagnosed as having a RET-associatedcancer (e.g., any of the RET-associated cancers described herein orknown in the art) (e.g., a subject diagnosed or identified as having aRET-associated cancer using any of the methods described herein or knownin the art), (ii) previously administered one or more doses (e.g., twoor more, three or more, four or more, five or more, or ten or more) ofthe compound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, as a monotherapy, and (ii) afteradministration of the one or more doses of the compound of Formula I-IV,or a pharmaceutically acceptable salt, amorphous, or polymorph formthereof, identified as having about the same or an elevated level ofcirculating tumor DNA as compared to a reference level of circulatingtumor DNA. In some embodiments of these methods, the reference level ofcirculating tumor DNA is a level of circulating tumor DNA in abiological sample (e.g., a biological sample comprising blood, serum, orplasma) obtained from the subject prior to administration of the one ormore doses of the compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof, as a monotherapy.Some embodiments further include determining the level of circulatingtumor DNA in the biological sample obtained from the subject prior toadministration of the one or more doses of the compound of Formula I-IV,or a pharmaceutically acceptable salt, amorphous, or polymorph formthereof, as a monotherapy. In some examples of these methods, thereference level of circulating tumor DNA is a threshold level ofcirculating tumor DNA (e.g., an average level of circulating tumor DNAin a population of subjects having a similar RET-associated cancer andhaving a similar stage of the RET-associated cancer, but receiving anon-effective treatment or a placebo, or not yet receiving therapeutictreatment, or a level of circulating tumor DNA in a subject having asimilar RET-associated cancer and having a similar stage of theRET-associated cancer, but receiving a non-effective treatment or aplacebo, or not yet receiving therapeutic treatment). In someembodiments of any of these methods, the additional therapeutic agent isa second RET kinase inhibitor (e.g., a second RET kinase inhibitorselected from the group of: cabozantinib, vandetanib, alectinib,apatinib, sitravatinib, sorafenib, lenvatinib, ponatinib, dovitinib,sunitinib, foretinib, BLU667, and BLU6864. In some embodiments of any ofthe methods described herein, the additional therapeutic agent ortreatment includes one or more of radiation therapy, a chemotherapeuticagent (e.g., any of the examples of a chemotherapeutic agent describedherein or known in the art), a checkpoint inhibitor (e.g., any of thecheckpoint inhibitors described herein or known in the art), surgery(e.g., at least partial resection of the tumor), and one or more otherkinase inhibitors (e.g., any of the other kinase inhibitors describedherein or known in the art).

Also provided herein are methods of determining the efficacy of atreatment in a subject that include: (a) determining a first level ofcirculating tumor DNA in a biological sample (e.g., a biological sampleincluding blood, serum, or plasma) obtained from a subject identified ordiagnosed as having a RET-associated cancer at a first time point; (b)administering a treatment including one or more doses of a compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof to the subject, after the first time point andbefore a second time point; (c) determining a second level ofcirculating tumor DNA in a biological sample (e.g., a biological samplecomprising blood, serum, or plasma) obtained from the subject at thesecond time point; and (d) identifying that the treatment is effectivein a subject determined to have a decreased second level of circulatingtumor DNA as compared to the first level of circulating tumor DNA; oridentifying the treatment is not effective in a subject determined tohave about the same or an elevated second level of circulating tumor DNAas compared to the first level of circulating tumor DNA. In someembodiments of these methods, the first time point and the second timepoint are about 1 week to about 1 year apart (e.g., about 1 week toabout 10 months, about 1 week to about 8 months, about 1 week to about 6months, about 1 week to about 4 months, about 1 week to about 3 months,about 1 week to about 2 months, about 1 week to about 1 month, or about1 week to about 2 weeks).

Also provided herein are methods of determining whether a subject hasdeveloped resistance to a treatment that include: (a) determining afirst level of circulating tumor DNA in a biological sample (e.g., abiological sample comprising blood, serum, or plasma) obtained from asubject identified or diagnosed as having a RET-associated cancer at afirst time point; (b) administering a treatment including one or more(e.g., two or more, three or more, four or more, five or more, or ten ormore) doses of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof to the subject,after the first time point and before a second time point; (c)determining a second level of circulating tumor DNA in a biologicalsample obtained from the subject at the second time point; and (d)determining that a subject having a decreased second level ofcirculating tumor DNA as compared to the first level of circulatingtumor DNA has not developed resistance to the treatment; or determiningthat a subject having about the same or an elevated second level ofcirculating tumor DNA as compared to the first level of circulatingtumor DNA has developed resistance to the treatment. In some embodimentsof these methods, the first time point and the second time point areabout 1 week to about 1 year apart (e.g., about 1 week to about 10months, about 1 week to about 8 months, about 1 week to about 6 months,about 1 week to about 4 months, about 1 week to about 3 months, about 1week to about 2 months, about 1 week to about 1 month, or about 1 weekto about 2 weeks).

Exemplary methods for detecting circulating tumor DNA are described inMoati et al., Clin. Res. Hepatol. Gastroenterol. Apr. 4, 2018; Oussalahet al., EBioMedicine Mar. 28, 2018; Moon et al., Adv. Drug Deliv. Rev.Apr. 4, 2018; Solassaol et al., Clin. Chem. Lab. Med. Apr. 7, 2018;Arriola et al., Clin. Transl. Oncol. Apr. 5, 2018; Song et al., J Circ.Biomark. Mar. 25, 2018; Aslibekyan et al., JAMA Cardiol. Apr. 4, 2018;Isbell et al., J. Thorac. Cardiovasc. Surg. Mar. 13, 2018; Boeckx etal., Clin. Colorectal Cancer Feb. 22, 2018; Anunobi et al., J Surg. Res.Mar. 28, 2018; Tan et al., Medicine 97(13):e0197, 2018; Reithdorf etal., Transl. Androl. Urol. 6(6):1090-1110, 2017; Volckmar et al., GenesChromosomes Cancer 57(3):123-139, 2018; and Lu et al., Chronic Dis.Transl. Med. 2(4):223-230, 2016. Additional methods for detectingcirculating tumor DNA are known in the art.

In some embodiments, provided herein are methods for treating aRET-associated cancer in a subject in need of such treatment, the methodcomprising (a) detecting a dysregulation of a RET gene, a RET kinase, orthe expression or activity or level of any of the same in a sample fromthe subject; and (b) administering to the subject a therapeuticallyeffective amount of a multikinase inhibitor, wherein the multikinaseinhibitor is selected from vandetanib or cabozantinib; or apharmaceutically acceptable salt or solvate thereof. In someembodiments, the methods further comprise (after (b)) (c) determiningwhether a cancer cell in a sample obtained from the subject has at leastone RET inhibitor resistance mutation; and (d) administering a compoundof Formula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof as a monotherapy or in conjunction with anotheranticancer agent to the subject if the subject has a cancer cell thathas at least one RET inhibitor resistance mutation; or (e) administeringadditional doses of the multikinase inhibitor of step (b) to the subjectif the subject has a cancer cell that does not have a RET inhibitorresistance mutation.

In some embodiments, provided herein are methods for treating aRET-associated cancer in a subject in need of such treatment, the methodcomprising (a) detecting a dysregulation of a RET gene, a RET kinase, orthe expression or activity or level of any of the same in a sample fromthe subject; and (b) administering to the subject a therapeuticallyeffective amount of a first multikinase inhibitor, wherein themulitkinase inhibitor is selected from the group consisting of:vandetanib or cabozantinib; or a pharmaceutically acceptable salt orsolvate thereof. In some embodiments, the methods further comprise(after (b)) (c) determining whether a cancer cell in a sample obtainedfrom the subject has at least one RET inhibitor resistance mutation; and(d) administering a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof as a monotherapyor in conjunction with another anticancer agent to the subject if thesubject has a cancer cell that has at least one RET inhibitor resistancemutation; or (e) administering additional doses of the multikinaseinhibitor of step (b) to the subject if the subject has a cancer cellthat does not have a RET inhibitor resistance mutation.

In some embodiments, provided herein are methods for treating aRET-associated cancer in a subject in need of such treatment, the methodcomprising (a) detecting one or more fusion proteins of Table 1 and/orone or more RET kinase protein point mutations/insertions/deletions ofTables 2 and 2a in a sample from the subject; and (b) administering tothe subject a therapeutically effective amount of a multikinaseinhibitor, wherein the multikinase inhibitor is selected from the groupconsisting of: vandetanib or cabozantinib; or a pharmaceuticallyacceptable salt or solvate thereof. In some embodiments, the methodsfurther comprise (after (b)) (c) determining whether a cancer cell in asample obtained from the subject has at least one RET inhibitorresistance mutation of Tables 3 or 4; and (d) administering a compoundof Formula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof as a monotherapy or in conjunction with anotheranticancer agent to the subject if the subject has a cancer cell thathas at least one RET inhibitor resistance mutation; or (e) administeringadditional doses of the multikinase inhibitor of step (b) to the subjectif the subject has a cancer cell that does not have a RET inhibitorresistance mutation.

In some embodiments, provided herein are methods for treating aRET-associated cancer in a subject in need of such treatment, the methodcomprising (a) detecting the fusion protein KIF5B-RET in a sample fromthe subject; and (b) administering to the subject a therapeuticallyeffective amount of a multikinase inhibitor, wherein the multikinaseinhibitor is selected from the group consisting of vandetanib orcabozantinib; or a pharmaceutically acceptable salt or solvate thereof.In some embodiments, the methods further comprise (after (b)) (c)determining whether a cancer cell in a sample obtained from the subjecthas the RET inhibitor resistance mutation V804M, G810S, or G810R; and(d) administering a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof selected from thegroup consisting of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof as a monotherapyor in conjunction with another anticancer agent to the subject if thesubject has a cancer cell that has at least one RET inhibitor resistancemutation; or (e) administering additional doses of the multikinaseinhibitor of step (b) to the subject if the subject has a cancer cellthat does not have a RET inhibitor resistance mutation.

As another example, provided herein are methods for treating aRET-associated cancer in a subject in need of such treatment, the methodcomprising (a) detecting a dysregulation of a RET gene, a RET kinase, orthe expression or activity or level of any of the same in a sample fromthe subject; and (b) administering to the subject a therapeuticallyeffective amount of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof. In someembodiments, the methods further comprise (after (b)) (c) determiningwhether a cancer cell in a sample obtained from the subject has at leastone RET inhibitor resistance mutation; and (d) administering amultikinase inhibitor (e.g., vandetanib or cabozantinib, as amonotherapy or in conjunction with another anticancer agent to thesubject if the subject has a cancer cell that has at least one RETinhibitor resistance mutation; or (e) administering additional doses ofthe compound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof of step (b) to the subject if thesubject has a cancer cell that does not have a RET inhibitor resistancemutation. In some embodiments, provided herein are methods for treatinga RET-associated cancer in a subject in need of such treatment, themethod comprising (a) detecting a dysregulation of a RET gene, a RETkinase, or the expression or activity or level of any of the same in asample from the subject; and (b) administering to the subject atherapeutically effective amount of a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof.In some embodiments, the methods further comprise (after (b)) (c)determining whether a cancer cell in a sample obtained from the subjecthas at least one RET inhibitor resistance mutation; and (d)administering a multikinase inhibitor (e.g., vandetanib orcabozantinib), as a monotherapy or in conjunction with anotheranticancer agent to the subject if the subject has a cancer cell thathas at least one RET inhibitor resistance mutation; or (e) administeringadditional doses of the compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof of step (b) to thesubject if the subject has a cancer cell that does not have a RETinhibitor resistance mutation. In some embodiments, provided herein aremethods for treating a RET-associated cancer in a subject in need ofsuch treatment, the method comprising (a) detecting one or more fusionproteins of Table 1 and/or one or more RET kinase protein pointmutations/insertions/deletions of Tables 2 and 2a in a sample from thesubject; and (b) administering to the subject a therapeuticallyeffective amount of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof. In someembodiments, the methods further comprise (after (b)) (c) determiningwhether a cancer cell in a sample obtained from the subject has at leastone RET inhibitor resistance mutation of Tables 3 or 4; and (d)administering a multikinase inhibitor (e.g., vandetanib orcabozantinib), as a monotherapy or in conjunction with anotheranticancer agent to the subject if the subject has a cancer cell thathas at least one RET inhibitor resistance mutation; or (e) administeringadditional doses of the compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof of step (b) to thesubject if the subject has a cancer cell that does not have a RETinhibitor resistance mutation. In some embodiments, provided herein aremethods for treating a RET-associated cancer in a subject in need ofsuch treatment, the method comprising (a) detecting the fusion proteinKIF5B-RET in a sample from the subject; and (b) administering to thesubject a therapeutically effective amount of a compound of FormulaI-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphform thereof. In some embodiments, the methods further comprise (after(b)) (c) determining whether a cancer cell in a sample obtained from thesubject has the RET inhibitor resistance mutation V804M, G810S, orG810R; and (d) administering a multikinase inhibitor (e.g., vandetanibor cabozantinib) as a monotherapy or in conjunction with anotheranticancer agent to the subject if the subject has a cancer cell thathas at least one RET inhibitor resistance mutation; or (e) administeringadditional doses of the compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof of step (b) to thesubject if the subject has a cancer cell that does not have a RETinhibitor resistance mutation.

Also, provided herein are methods for treating a RET-associated cancerin a subject in need of such treatment, the method comprising (a)detecting a dysregulation of a RET gene, a RET kinase, or the expressionor activity or level of any of the same in a sample from the subject;and (b) administering to the subject a therapeutically effective amountof a compound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof. In some embodiments, the methodsfurther comprise (after (b)) (c) determining whether a cancer cell in asample obtained from the subject has at least one RET inhibitorresistance mutation; and (d) administering additional doses of thecompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof of step (b) to the subject as amonotherapy or in conjunction with another anticancer agent (e.g., asecond RET inhibitor, a second compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,or immunotherapy) or anticancer therapy (e.g., surgery or radiation) ifthe subject has a cancer cell that has at least one RET inhibitorresistance mutation. In some embodiments, provided herein are methodsfor treating a RET-associated cancer in a subject in need of suchtreatment, the method comprising (a) detecting a dysregulation of a RETgene, a RET kinase, or the expression or activity or level of any of thesame in a sample from the subject; and (b) administering to the subjecta therapeutically effective amount of a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof.In some embodiments, the methods further comprise (after (b)) (c)determining whether a cancer cell in a sample obtained from the subjecthas at least one RET inhibitor resistance mutation; and (d)administering additional doses of the compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofof step (b) to the subject as a monotherapy or in conjunction withanother anticancer agent (e.g., a second RET inhibitor, a secondcompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, or immunotherapy) or anticancertherapy (e.g., surgery or radiation) if the subject has a cancer cellthat has at least one RET inhibitor resistance mutation. In someembodiments, provided herein are methods for treating a RET-associatedcancer in a subject in need of such treatment, the method comprising (a)detecting one or more fusion proteins of Table 1 and/or one or more RETkinase protein point mutations/insertions/deletions of Tables 2 and 2ain a sample from the subject; and (b) administering to the subject atherapeutically effective amount of a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofselected from the group consisting of a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof.In some embodiments, the methods further comprise (after (b)) (c)determining whether a cancer cell in a sample obtained from the subjecthas at least one RET inhibitor resistance mutation of Tables 3 or 4; and(d) administering additional doses of the compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofof step (b) to the subject as a monotherapy or in conjunction withanother anticancer agent (e.g., a second RET inhibitor, a secondcompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, or immunotherapy) or anticancertherapy (e.g., surgery or radiation) if the subject has a cancer cellthat has at least one RET inhibitor resistance mutation. In someembodiments, a second RET inhibitor selected from the group consistingof alectinib, cabozantinib, lenvatinib, nintedanib, ponatinib,regorfenib, sorafenib, sunitinib, vandetanib, RXDX-105 (agerafenib),BLU-667 ((1S,4R)—N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methoxy-4-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide),BLU6864, DS-5010, GSK3179106, GSK3352589, and NMS-E668 is administeredin step (d). In some embodiments, provided herein are methods fortreating a RET-associated cancer in a subject in need of such treatment,the method comprising (a) detecting the fusion protein KIF5B-RET in asample from the subject; and (b) administering to the subject atherapeutically effective amount of a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof.In some embodiments, the methods further comprise (after (b)) (c)determining whether a cancer cell in a sample obtained from the subjecthas the RET inhibitor resistance mutation V804M, G810S, or G810R; and(d) administering additional doses of the compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofof step (b) to the subject as a monotherapy or in conjunction withanother anticancer agent (e.g., a second RET inhibitor, a secondcompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, or immunotherapy) or anticancertherapy (e.g., surgery or radiation) if the subject has a cancer cellthat has at least one RET inhibitor resistance mutation. In someembodiments, a second RET inhibitor selected from the group consistingof alectinib, cabozantinib, lenvatinib, nintedanib, ponatinib,regorfenib, sorafenib, sunitinib, vandetanib, RXDX-105 (agerafenib),BLU-667 ((1S,4R)—N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methoxy-4-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide),BLU6864, DS-5010, GSK3179106, GSK3352589, and NMS-E668 is administeredin step (d).

Also, provided herein are methods for treating a RET-associated cancerin a subject in need of such treatment, the method comprising (a)detecting a dysregulation of a RET gene, a RET kinase, or the expressionor activity or level of any of the same in a sample from the subject;and (b) administering to the subject a therapeutically effective amountof a compound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof. In some embodiments, the methodsfurther comprise (after (b)) (c) detecting at least one RET inhibitorresistance mutation in a cancer cell in a sample obtained from thesubject; and (d) administering additional doses of the compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof of step (b) to the subject as a monotherapy or inconjunction with another anticancer agent (e.g., a second RET inhibitor,a second compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof, or immunotherapy) oranticancer therapy (e.g., surgery or radiation). In some embodiments,provided herein are methods for treating a RET-associated cancer in asubject in need of such treatment, the method comprising (a) detecting adysregulation of a RET gene, a RET kinase, or the expression or activityor level of any of the same in a sample from the subject; and (b)administering to the subject a therapeutically effective amount of acompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof. In some embodiments, the methodsfurther comprise (after (b)) (c) detecting at least one RET inhibitorresistance mutation in a cancer cell in a sample obtained from thesubject; and (d) administering additional doses of the compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof of step (b) to the subject as a monotherapy or inconjunction with another anticancer agent (e.g., a second RET inhibitor,a second compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof, or immunotherapy) oranticancer therapy (e.g., surgery or radiation). In some embodiments,provided herein are methods for treating a RET-associated cancer in asubject in need of such treatment, the method comprising (a) detectingone or more fusion proteins of Table 1 and/or one or more RET kinaseprotein point mutations/insertions/deletions of Tables 2 and 2a in asample from the subject; and (b) administering to the subject atherapeutically effective amount of a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofselected from the group consisting of a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof.In some embodiments, the methods further comprise (after (b)) (c)detecting at least one RET inhibitor resistance mutation of Tables 3 or4 in a cancer cell in a sample obtained from the subject; and (d)administering additional doses of the compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofof step (b) to the subject as a monotherapy or in conjunction withanother anticancer agent (e.g., a second RET inhibitor, a secondcompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, or immunotherapy) or anticancertherapy (e.g., surgery or radiation). In some embodiments, a second RETinhibitor selected from the group consisting of alectinib, cabozantinib,lenvatinib, nintedanib, ponatinib, regorfenib, sorafenib, sunitinib,vandetanib, RXDX-105 (agerafenib), BLU-667 ((1S,4R)—N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methoxy-4-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide),BLU6864, DS-5010, GSK3179106, GSK3352589, and NMS-E668 is administeredin step (d). In some embodiments, provided herein are methods fortreating a RET-associated cancer in a subject in need of such treatment,the method comprising (a) detecting the fusion protein KIF5B-RET in asample from the subject; and (b) administering to the subject atherapeutically effective amount of a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof.In some embodiments, the methods further comprise (after (b)) (c)detecting the RET inhibitor resistance mutation V804M, G810S, or G810Rin a cancer cell in a sample obtained from the subject; and (d)administering additional doses of the compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofof step (b) to the subject as a monotherapy or in conjunction withanother anticancer agent (e.g., a second RET inhibitor, a secondcompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, or immunotherapy) or anticancertherapy (e.g., surgery or radiation). In some embodiments, a second RETinhibitor selected from the group consisting of alectinib, cabozantinib,lenvatinib, nintedanib, ponatinib, regorfenib, sorafenib, sunitinib,vandetanib, RXDX-105 (agerafenib), BLU-667((1S,4R)—N—((S)-1-(6-(4-fluoro-1H-pyrazol-1-yl)pyridin-3-yl)ethyl)-1-methoxy-4-(4-methyl-6-((5-methyl-1H-pyrazol-3-yl)amino)pyrimidin-2-yl)cyclohexane-1-carboxamide),BLU6864, DS-5010, GSK3179106, GSK3352589, and NMS-E668 is administeredin step (d).

Further provided herein is a method for treating lung cancer in apatient in need thereof, the method comprising administering to thepatient a therapeutically effective amount of a compound of FormulaI-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphform thereof, crizotinib, osimertinib, or any combination thereof.

In some embodiments, the lung cancer is a RET-associated cancer. Forexample, the method can include: (a) detecting a dysregulation of a RETgene, a RET kinase, or the expression or activity or level of any of thesame in a sample from the subject; and (b) administering to the subjecta therapeutically effective amount of a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof.In some embodiments, the methods further comprises (after (b)) (c)determining whether a cancer cell in a sample obtained from the subjecthas at least one RET inhibitor resistance mutation (e.g., a METdysregulation such as a MET gene amplification); and (d) administering asecond therapeutic agent, wherein the second therapeutic agent iscrizotinib, as a monotherapy or in conjunction with a compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof to the subject if the subject has a cancer cellthat has at least one RET inhibitor resistance mutation; or (e)administering additional doses of the compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofof step (b) to the subject if the subject has a cancer cell that doesnot have a RET inhibitor resistance mutation. In some such embodiments,the method comprises (a) detecting one or more fusion proteins of Table1 and/or one or more RET kinase protein point mutations/insertions ofTable 2 in a sample from the subject; and (b) administering to thesubject a therapeutically effective amount of a compound of FormulaI-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphform thereof. In further embodiments, the methods further comprise(after (b)) (c) determining whether a cancer cell in a sample obtainedfrom the subject has at least one RET inhibitor resistance mutation(e.g., a MET dysregulation such as a MET gene amplification); and (d)administering a second therapeutic agent, wherein the second therapeuticagent is crizotinib, as a monotherapy or in conjunction with a compoundof Formula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof to the subject if the subject has a cancer cellthat has at least one RET inhibitor resistance mutation; or (e)administering additional doses of the compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofof step (b) to the subject if the subject has a cancer cell that doesnot have a RET inhibitor resistance mutation.

In some embodiments, the lung cancer is an EGFR-associated cancer. Forexample, the method can include: (a) detecting a dysregulation of anEGFR gene, an EGFR kinase, or the expression or activity or level of anyof the same in a sample from the subject; and (b) administering to thesubject a therapeutically effective amount of an EGFR inhibitor (e.g.,osimertinib). In some embodiments, the methods further comprises (after(b)) (c) determining whether a cancer cell in a sample obtained from thesubject has at least one dysregulation of a RET gene, a RET kinase, orthe expression or activity or level of any of the same (e.g., a RET genefusion); and (d) administering a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,as a monotherapy or in conjunction with the EGFR inhibitor (e.g.,osimertinib) to the subject if the subject has a cancer cell that has atleast one dysregulation of a RET gene, a RET kinase, or the expressionor activity or level of any of the same (e.g., a RET gene fusion); or(e) administering additional doses of the EGFR inhibitor (e.g.,osimertinib) of step (b) to the subject if the subject has a cancer cellthat does not have a dysregulation of a RET gene, a RET kinase, or theexpression or activity or level of any of the same (e.g., a RET genefusion). In some such embodiments, the method comprises (a) detecting adysregulation of an EGFR gene, an EGFR kinase, or the expression oractivity or level of any of the same in a sample from the subject; and(b) administering to the subject a therapeutically effective amount ofosimertinib. In further embodiments, the methods further comprise (after(b)) (c) determining whether a cancer cell in a sample obtained from thesubject has one or more fusion proteins of Table 1 and/or one or moreRET kinase protein point mutations/insertions of Table 2; and (d)administering a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof, as a monotherapyor in conjunction with osimertinib to the subject if the subject has acancer cell that has one or more fusion proteins of Table 1 and/or oneor more RET kinase protein point mutations/insertions of Table 2; or (e)administering additional doses of the osimertinib of step (b) to thesubject if the subject has a cancer cell that does not have one or morefusion proteins of Table 1 and/or one or more RET kinase protein pointmutations/insertions of Table 2.

The term “EGFR-associated cancer” as used herein refers to cancersassociated with or having a dysregulation of a EGFR gene, a EGFR kinase,or expression or activity, or level of any of the same.

The phrase “dysregulation of a EGFR gene, a EGFR kinase, or theexpression or activity or level of any of the same” refers to a geneticmutation (e.g., a EGFR gene translocation that results in the expressionof a fusion protein, a deletion in a EGFR gene that results in theexpression of a EGFR protein that includes a deletion of at least oneamino acid as compared to the wild-type EGFR protein, or a mutation in aEGFR gene that results in the expression of a EGFR protein with one ormore point mutations, or an alternative spliced version of a EGFR mRNAthat results in a EGFR protein that results in the deletion of at leastone amino acid in the EGFR protein as compared to the wild-type EGFRprotein), or a EGFR gene amplification that results in overexpression ofa EGFR protein or an autocrine activity resulting from theoverexpression of a EGFR gene a cell, that results in a pathogenicincrease in the activity of a kinase domain of a EGFR protein (e.g., aconstitutively active kinase domain of a EGFR protein) in a cell. Asanother example, a dysregulation of a EGFR gene, a EGFR protein, orexpression or activity, or level of any of the same, can be a mutationin a EGFR gene that encodes a EGFR protein that is constitutively activeor has increased activity as compared to a protein encoded by a EGFRgene that does not include the mutation. For example, a dysregulation ofa EGFR gene, a EGFR protein, or expression or activity, or level of anyof the same, can be the result of a gene or chromosome translocationwhich results in the expression of a fusion protein that contains afirst portion of EGFR that includes a functional kinase domain, and asecond portion of a partner protein (i.e., that is not EGFR). In someexamples, dysregulation of a EGFR gene, a EGFR protein, or expression oractivity, can be a result of a gene translocation of one EGFR gene withanother non-EGFR gene. In some embodiments, the EGFR mutation is a T790Mmutation. In some embodiments, the EGFR mutation is a C797S mutation.

The term “wildtype EGFR” or “wild-type EGFR” describes a nucleic acid(e.g., a EGFR gene or a EGFR mRNA) or protein (e.g., a EGFR protein)that is found in a subject that does not have a EGFR-associated cancer(and optionally also does not have an increased risk of developing aEGFR-associated cancer and/or is not suspected of having aEGFR-associated cancer), or is found in a cell or tissue from a subjectthat does not have a EGFR-associated cancer (and optionally also doesnot have an increased risk of developing a EGFR-associated cancer and/oris not suspected of having a EGFR-associated cancer).

Also provided are methods of selecting a treatment for a subject havinga cancer that include: identifying a subject having a cancer cell thathas one or more RET inhibitor resistance mutations; and selecting atreatment that includes administration of a compound of Formula I-IV, ora pharmaceutically acceptable salt, amorphous, or polymorph formthereof. In some embodiments, the one or more RET inhibitor resistancemutations confer increased resistance to a cancer cell or tumor totreatment with a first RET inhibitor. In some embodiments, the compoundof Formula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof is administered in combination with the first RETinhibitor. Also provided are methods of selecting a treatment for asubject having a cancer that include: selecting a treatment thatincludes administration of a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereoffor a subject identified as having a cancer cell that has one or moreRET inhibitor resistance mutations. Also provided are methods ofselecting a subject having a cancer for a treatment that does notinclude a first RET inhibitor as a monotherapy that include: identifyinga subject having a cancer cell that has one or more RET inhibitorresistance mutations; and selecting the identified subject for atreatment that includes a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof.Also provided are methods of selecting a subject having a cancer for atreatment that does not include a first RET inhibitor as a monotherapythat include: selecting a subject identified as having a cancer cellthat has one or more RET inhibitor resistance mutations for a treatmentthat includes administration of a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof.In some embodiments, the one or more RET inhibitor resistance mutationsinclude one or more RET inhibitor resistance mutations listed in Tables3 and 4. In some embodiments, the one or more RET inhibitor resistancemutations can include a substitution at amino acid position 804, e.g.,V804M, V804L, or V804E, or a substitution amino acid position 810, e.g.,G810S, G810R, G810C, G810A, G810V, and G810D.

Also provided are methods of determining the likelihood that a subjecthaving a cancer (e.g., a RET-associated cancer) will have a positiveresponse to treatment with a first RET inhibitor as a monotherapy thatinclude: determining whether a cancer cell in a sample obtained from thesubject has one or more RET inhibitor resistance mutations; anddetermining that a subject having a cancer cell that has one or more RETinhibitor resistance mutations has a decreased likelihood of having apositive response (i.e. an increased likelihood of having a negativeresponse) to treatment with a first RET inhibitor as a monotherapy. Alsoprovided are methods of determining the likelihood that a subject havinga cancer (e.g., a RET-associated cancer) will have a positive responseto treatment with a first RET inhibitor as a monotherapy that include:determining whether a cancer cell in a sample obtained from the subjecthas one or more RET inhibitor resistance mutations; and determining thata subject not having a cancer cell that has one or more RET inhibitorresistance mutations has an increased likelihood of having a positiveresponse to treatment with a first RET inhibitor as a monotherapy ascompared to a subject having a cancer cell that has one or more RETinhibitor resistance mutations. Also provided are methods of predictingthe efficacy of treatment with a first RET inhibitor as a monotherapy ina subject having cancer that include: determining whether a cancer cellin a sample obtained from the subject has one or more RET inhibitorresistance mutations; and determining that treatment with a first RETinhibitor as a monotherapy is less likely to be effective in a subjecthaving a cancer cell in a sample obtained from the subject that has oneor more RET inhibitor resistance mutations. Also provided are methods ofpredicting the efficacy of treatment with a first RET inhibitor as amonotherapy in a subject having cancer that include: determining thattreatment with a first RET inhibitor as a monotherapy is less likely tobe effective in a subject having a cancer cell in a sample obtained fromthe subject that has one or more RET inhibitor resistance mutations. Insome embodiments, the one or more RET inhibitor resistance mutationsconfer increased resistance to a cancer cell or tumor to treatment withthe first RET inhibitor. In some embodiments, the one or more RETinhibitor resistance mutations include one or more RET inhibitorresistance mutations listed in Tables 3 and 4. For example, the one ormore RET inhibitor resistance mutations can include a substitution atamino acid position 804, e.g., V804M, V804L, or V804E, or a substitutionat amino acid position 810, e.g., G810S, G810R, G810C, G810A, G810V, andG810D.

Also provided are methods of treating a subject having a cancer thatinclude: (a) administering one or more doses of a first RET inhibitor tothe subject for a period of time; (b) after (a), determining whether acancer cell in a sample obtained from the subject has at least one RETinhibitor resistance mutation; and (c) administering a compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof as a monotherapy or in conjunction with anotheranticancer agent to the subject if the subject has a cancer cell thathas at least one RET inhibitor resistance mutation; or (d) administeringadditional doses of the first RET inhibitor of step (a) to the subjectif the subject has a cancer cell that does not have a RET inhibitorresistance mutation. In some embodiments, where the subject isadministered additional doses of the first RET inhibitor of step (a),the subject can also be administered another anticancer agent (e.g., asecond RET inhibitor or a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,or immunotherapy). In some embodiments, the additional anticancer agentis any anticancer agent known in the art. For example, the additionalanticancer agent is another RET inhibitor (e.g., a second RETinhibitor). In some embodiments, the additional anticancer agent is animmunotherapy. In some embodiments of step (c), another RET inhibitorcan be the first RET inhibitor administered in step (a). In someembodiments, the one or more RET inhibitor resistance mutations conferincreased resistance to a cancer cell or tumor to treatment with thefirst RET inhibitor. In some embodiments, the one or more RET inhibitorresistance mutations include one or more RET inhibitor resistancemutations listed in Tables 3 and 4. For example, the one or more RETinhibitor resistance mutations can include a substitution at amino acidposition 804, e.g., V804M, V804L, or V804E, or a substitution at aminoacid position 810, e.g., G810S, G810R, G810C, G810A, G810V, and G810D.

Also provided are methods of treating a subject having a cancer thatinclude: (a) administering one or more doses of a first RET inhibitor tothe subject for a period of time; (b) after (a), determining whether acancer cell in a sample obtained from the subject has at least one RETinhibitor resistance mutation; and (c) administering a second RETinhibitor as a monotherapy or in conjunction with another anticanceragent to the subject if the subject has a cancer cell that has at leastone RET inhibitor resistance mutation; or (d) administering additionaldoses of the first RET inhibitor step (a) to the subject if the subjecthas a cancer cell that does not have a RET inhibitor resistancemutation. In some embodiments, where the subject is administeredadditional doses of the first RET inhibitor of step (a), the subject canalso be administered another anticancer agent. In some embodiments, theone or more RET inhibitor resistance mutations confer increasedresistance to a cancer cell or tumor to treatment with the first RETinhibitor. In some embodiments, the one or more RET inhibitor resistancemutations include one or more RET inhibitor resistance mutations listedin Tables 3 and 4. For example, the one or more RET inhibitor resistancemutations can include a substitution at amino acid position 804, e.g.,V804M, V804L, or V804E, or a substitution at amino acid position 810,e.g., G810S, G810R, G810C, G810A, G810V, and G810D. In some embodiments,the additional anticancer agent is any anticancer agent known in theart. For example, the additional anticancer agent is another RETinhibitor (e.g., a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof). In someembodiments, the additional anticancer agent is an immunotherapy.

Also provided are methods of treating a subject having a cancer (e.g., aRET-associated cancer) that include: (a) determining whether a cancercell in a sample obtained from a subject having a cancer and previouslyadministered one or more doses of a first RET inhibitor, has one or moreRET inhibitor resistance mutations; and (b) administering a compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof as a monotherapy or in conjunction with anotheranticancer agent to the subject if the subject has a cancer cell thathas at least one RET inhibitor resistance mutation; or (c) administeringadditional doses of the first RET inhibitor previously administered tothe subject if the subject has cancer cell that does not have a RETinhibitor resistance mutation. In some embodiments, where the subject isadministered additional doses of the first RET inhibitor previouslyadministered to the subject, the subject can also be administeredanother anticancer agent (e.g., a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,or immunotherapy). In some embodiments, the one or more RET inhibitorresistance mutations confer increased resistance to a cancer cell ortumor to treatment with the first RET inhibitor. In some embodiments,the one or more RET inhibitor resistance mutations include one or moreRET inhibitor resistance mutations listed in Tables 3 and 4. Forexample, the one or more RET inhibitor resistance mutations can includea substitution at amino acid position 804, e.g., V804M, V804L, or V804E,or a substitution at amino acid position 810, e.g., G810S, G810R, G810C,G810A, G810V, and G810D. In some embodiments, the additional anticanceragent is any anticancer agent known in the art. For example, theadditional anticancer agent is another RET inhibitor (e.g., a second RETinhibitor). In some embodiments, the additional anticancer agent is animmunotherapy. In some embodiments of step (b), another anticancer agentcan be the first RET inhibitor administered in step (a).

Also provided are methods of treating a subject having a cancer thatinclude: (a) determining whether a cancer cell in a sample obtained froma subject having a cancer and previously administered one or more dosesof a first RET inhibitor has one or more RET inhibitor resistancemutations; and (b) administering a second RET inhibitor as a monotherapyor in conjunction with another anticancer agent to the subject if thesubject has a cancer cell that has at least one RET inhibitor resistancemutation; or (c) administering additional doses of the first RETinhibitor previously administered to the subject if the subject has acancer cell that does not have a RET inhibitor resistance mutation. Insome embodiments, where the subject is administered additional doses ofthe first RET inhibitor previously administered to the subject, thesubject can also be administered another anticancer agent. In someembodiments, the one or more RET inhibitor resistance mutations conferincreased resistance to a cancer cell or tumor to treatment with thefirst RET inhibitor. In some embodiments, the one or more RET inhibitorresistance mutations include one or more RET inhibitor resistancemutations listed in Tables 3 and 4. For example, the one or more RETinhibitor resistance mutations can include a substitution at amino acidposition 804, e.g., V804M, V804L, or V804E, or a substitution at aminoacid position 810, e.g., G810S, G810R, G810C, G810A, G810V, and G810D.In some embodiments, the additional anticancer agent is any anticanceragent known in the art. For example, the additional anticancer agent isanother RET inhibitor (e.g., a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof).In some embodiments, the additional anticancer agent is animmunotherapy. In some embodiments of (b), another anticancer agent canbe the first RET inhibitor administered in step (a).

In some embodiments, a RET-associated cancer as described herein canoccur in a subject along with a dysregulation of another gene, anotherprotein, or the expression or activity or level of any of the same.

For example, a RET-associated cancer that exhibits a RET fusion canoccur in a subject along with one or more of: a dysregulation of a METgene, a MET protein, or the expression or activity or level of any ofthe same; a dysregulation of a PIK3CA gene, a PIK3CA protein, or theexpression or activity or level of any of the same; a dysregulation of aKRAS gene, a KRAS protein, or the expression or activity or level of anyof the same; a dysregulation of a EGFR gene, a EGFR protein, or theexpression or activity or level of any of the same (e.g., anamplification of a EGFR gene); a dysregulation of a FGFR2 gene, a FGFR2protein, or the expression or activity or level of any of the same (eg.,a fusion of an FGFR2 gene or an FGFR2 protein); a dysregulation of aCDK4 gene, a CDK4 protein, or the expression or activity or level of anyof the same (e.g., an amplification of a CDK4 gene); a dysregulation ofa mTOR gene, a mTOR protein, or the expression or activity or level ofany of the same; a dysregulation of a CDKN2A gene, a CDKN2A protein, orthe expression or activity or level of any of the same (e.g., a deletionin a CDKN2A gene or a CDKN2A protein); a dysregulation of a CDKN2B gene,a CDKN2B protein, or the expression or activity or level of any of thesame (e.g., a deletion in a CDKN2B gene or a CDKN2B protein); adysregulation of a NF1 gene, a NF1 protein, or the expression oractivity or level of any of the same; a dysregulation of a MYC gene, aMYC protein, or the expression or activity or level of any of the same(e.g., an amplification in a MYC gene); a dysregulation of a MDM2 gene,a MDM2 protein, or the expression or activity or level of any of thesame (e.g., an amplification in a MDM2 gene); a dysregulation of a GNASgene, a GNAS protein, or the expression or activity or level of any ofthe same; a dysregulation of a BRCA2 gene, a BRCA2 protein, or theexpression or activity or level of any of the same.

In some embodiments, a RET-associated cancer that exhibits a mutation ofa RET gene and/or a RET protein can occur in a subject along with one ormore of: a dysregulation of a PIK3CA gene, a PIK3CA protein, or theexpression or activity or level of any of the same; a dysregulation of aKRAS gene, a KRAS protein, or the expression or activity or level of anyof the same; a dysregulation of a EGFR gene, a EGFR protein, or theexpression or activity or level of any of the same; a dysregulation of aFGFR1 gene, a FGFR1 protein, or the expression or activity or level ofany of the same (e.g, an amplification of a FGFR1 gene); a dysregulationof a FGFR2 gene, a FGFR2 protein, or the expression or activity or levelof any of the same (e.g., an amplification of a FGFR2 gene); adysregulation of a FGFR3 gene, a FGFR3 protein, or the expression oractivity or level of any of the same (e.g., a fusion of a FGFR3 gene ora FGFR3 protein); a dysregulation of a ERBB2 gene, a ERBB2 protein, orthe expression or activity or level of any of the same (e.g., anamplification of ERBB2 gene); and a dysregulation of a KIT gene, a KITprotein, or the expression or activity or level of any of the same.

In some embodiments, a RET-associated cancer that exhibits anamplification of a RET gene can occur in a patient along with one ormore additional kinase amplifications. For example, am amplification ina FGFR1 gene; an amplification in a FGFR2 gene; an amplification in aFGFR3 gene; an amplification of a FGFR4 gene; an amplification of a CDK4gene; and an amplification in a CDK6 gene.

In some embodiments, wherein a RET-associated cancer as described hereincan occur in a subject along with a dysregulation in another kinase, themethods described herein can further comprise administration of anadditional therapeutic agent that targets and/or treats thedysregulation in the other kinase. For example, provided herein aremethods for treating a RET-associated cancer in a subject in need ofsuch treatment, the method comprising (a) detecting a dysregulation of aRET gene, a RET kinase, or the expression or activity or level of any ofthe same in a sample from the subject; and (b) administering to thesubject a therapeutically effective amount of a compound of FormulaI-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphform thereof. In some embodiments, the method further comprises (c)detecting a dysregulation in another kinase in a sample from thesubject; and (d) administering to the subject a therapeutic agent thattargets and/or treats the dysregulation in the other kinase. In someembodiments, the administration of a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofis done concurrently, sequentially, or serially. In some embodiments,the detecting steps (a) and (c) can be done simultaneously orsequentially in any order.

Additional therapeutic agents that target and/or treat the dysregulationof the other kinase can include any known inhibitor of the other kinase.Examples of such agents are as follows:

Exemplary PARP inhibitors include: 3-aminobenzamide (INO-1001),5-aminoisoquinoline, ABT472, ABT767, AG140361, AG14032, ANG2864,ANG3186, AZD2281, AZD2461, BGP-15, BSI101, BSI401, CEP6800, CEP8983,CK102, CEP9722 (prodrug of CEP8983), CPH101 with CPH102, DR2313, E7016(GPI-21016), E7449, GP16150, IMP4297, IMP04149, INO1002, INO1003,JPI283, JPI289, KU0687, KU58948, niraparib (MK-4827), NT125, olaparib(AZD2281), ONO-1924H, ONO2231, pamiparib (BGB-290), PJ-34, rucaparib(AG014699), SC10914, SOMCL9112, talazoparib (BMN-673), and veliparib(ABT-888).

Exemplary CDK 4/6 inhibitors include: palbociclib (PD0332991),abemaciclib (LY2835219), ribociclib (LEE011), trilaciclib (G1T28),voruciclib, and G1T38.

Exemplary ERBB2 (HER2/neu) inhibitors include: afatinib, afatinib,dacomitinib (PF-00299804), DS8201-a, erlontinib, gefitinib, KU004,lapatinib, laptinib ditosylate, MM-111, mubritinib (TAK-165), neratinib,pyrotinib (HTI-1001), tucatinib (ONT-380, ARRY-380), 7C3, cetuximab,HER2-BsAb, hersintuzumab, margetuximab, MI130004, NeuVax, paitumumab,pertuzumab, SYD985, trastuzumab, and trastuzumab emtansine.

Exemplary inhibitors of amplified ERBB2 (HER2/neu) include dacomitinib(PF-00299804), lapatinib, neratinib, pertuzumab, trastuzumab, andtrastuzumab emtansine.

Exemplary EGFR inhibitors include: AC0010, afatinib, AP26113, ASP8273,avatinib, avitinib, AZD3759, BMS-690514, brigatinib, canertinib,Cap-701, CHMFL-EGFR-202, CUDC-101, dacomitinib, EAI045, EGF816,erlontinib, erlotinib, gefitinib, GNS-1481, GNS-1486, Gö6976, HS-10296,icotinib, KU004, lapatinib, nazartinib, neratinib, olmutinib (HM61713,BI 1482694), osimertinib, osimertinib (AZD9291), pelitinib, PF-06747775,PKC412, pyrotinib (HTI-1001), rocilentinib, vandetanib, varlitinib,XL647, 7C3, cetuximab, depatuxizumab mafodotin (ABT-414), matuzumab,nimotuzumab, panitumumab, and zalutumumab.

Exemplary wild-type EGFR inhibitors include: afatinib, BMS-690514,canertinib, CUDC-101, dacomitinib, erlotinib, gefitinib, lapatinib,neratinib, pelitinib, vandetanib, varlitinib, XL647, cetuximab,matuzumab, nimotuzumab, panitumumab, and zalutumumab.

Exemplary inhibitors of mutated EGFR include: AC0010, afatinib, AP26113,ASP8273, avatinib, avitinib, AZD3759, BMS-690514, brigatinib,canertinib, Cap-701, CHMFL-EGFR-202, CUDC-101, dacomitinib, EAI045,EGF816, GNS-1481, GNS-1486, Gö6976, HS-10296, icotinib, nazartinib,neratinib, olmutinib (HM61713, BI 1482694), osimertinib (AZD9291),PF-06747775, PKC412, rocilentinib, vandetanib, varlitinib, andcetuximab.

An exemplary inhibitor of amplified EGFR is depatuxizumab mafodotin(ABT-414).

Exemplary inhibitors of FGFR include: ASP5878, AZD4547, BGJ398, BLU9931,brivatinib, cediranib, DEBIO 1347, derazantinib (ARQ-087), dovitinib(CHIR258), E7090, ENMD-2076, erdafitinib (JNJ-42756293), FGF 401,FIIN-1, FRIN-1, INCB054828, L16H50, lenvatinib, lucitanib, LY2874455,nintedanib, NP603, orantinib (SU6668), pazopanib, PBI05204, PD173074,ponatinib, PRN1371, regorafenib, rogaratinib (BAY-1163877), 549076,SOMCL-085, SU5402, sunitinib, TAS-120, FP-1039, GAL-F2, GAL-FR21,GAL-FR22, GAL-FR23, GP369, hLD1.vb, LD1, MFGR1877S, MM-161, PRO-001, andR3Mab.

Exemplary inhibitors of FGFR fusions include: BGJ398, DEBIO 1347,derazantinib (ARQ-087), E7090, erdafitinib (JNJ-42756293), lucitanib,and TAS-120.

Exemplary inhibitors of FGFR1, FGFR2, and FGFR3 include: AZD4547,BGJ398, DEBIO 1347, E7090, INCB054828, 549076, SOMCL-085, and TAS-120.

Exemplary inhibitors of FGF4 include: BLU-554, BLU9931, NVP-FGF401, andhLD1.vb.

Exemplary inhibitors of amplified FGFR1 include: AZD4547, BGJ398, DEBIO1347, derazantinib (ARQ-087), erdafitinib (JNJ-42756293), INCB054828,and lucitanib.

Exemplary inhibitors of amplified FGFR2 include: AZD4547, DEBIO 1347,derazantinib (ARQ-087), lucitanib, regorafenib, and TAS-120.

An exemplary inhibitor of amplified FGFR3 is AZD4547.

Exemplary MEK inhibitors include: AZD8330 (ARRY-424704), AZD6244(ARRY-142866), BI-847325, binimetinib, BIX02188, BIX02189, CH4987655,CH5126766, CI-1040, cobemetinib (GDC-0973), EBI-1051, G-573, G8935,GDC-0623, Myricetin, nobiletin, PD0325901, PD184161, PD318088, PD98059,PD334581, pimasertib (AS-703026), refametinib (RDEA119, BAY 869766),selumentinib (AZD6244), SL-327, TAK-733, trametinib, and U0126.

Exemplary KRAS inhibitors include: 0375-0604, a covalentquinazoline-based switch II pocket (SIIP) compound, ARS-1620, AZD4785,and LP1.

Exemplary PI3K inhibitors include: 3-methyladenine, A66, alpelisib(BYL719), AMG319, apitolisib (GDC-0980, RG7422), AS-252424, AS-604850,AS-605240, AZD6842, AZD8186, AZD8835, BGT226 (NVP-BGT226), buparlisib(BKM120), CAY10505, CH5132799, copanlisib (BAY 80-6946), CUDC-907,CZC24832, dactolisib (BEZ235, NVP-BEZ235), DS7423, duvelisib (IPI-145,INK1197), GDC-0032, GDC-0084, GDC-0326, gedatolisib (PF-05212384,PKI-5587), GNE-317, GS-9820, GSK1059615, GSK2292767, GSK2636771, HS-173,IC-87114, Idelalisib (CAL-101, GS-1101), IPI-145, IPI-3063, IPI-549,LY294002, LY3023414, nemiralisib (GSK2269557), omipalisib (GSK2126458,GSK458), PF-04691502, PF-4989216, PI-103, PI-3065, pictilisib(GDC-0941), PIK-293, PIK-294, PIK-75, PIK-90, PIK-93, PIK-III,pilaralisib (XL147), PKI-587, PP-110, PQR309, PQR309, PW-12, PX-866,quercetin, 514161, SAR245409 (XL765), SAR260301, SAR405, serabelisib(INK-1117, MLN-1117, TAK-1117), SF-1126, SF-2523, SN32976, taselisib(GDC-0032), TB101110, TG100-115, TG100-713, TGR-1202, TGX-221,umbralisib (TGR-1202), voxtalisib (XL765, SAR245409), VPS34-IN1, VS-5584(SB2343), WJDO08, wortmannin, and ZSTK474.

Exemplary KIT inhibitors include: AMG 706, amuvatinib (MP-470), APcK110,axitinib (AG-013736), AZD2932, dasatinib (BMS-354825), dovitinib(TKI-258, CHIR-258), EXEL-0862, imatinib, KI-328, masitinib (AB1010),midostaurin, MLN518, motesanib,N3-(6-aminopyridin-3-yl)-N1-(2-cyclopentylethyl)-4-methylisophthalamide,nilotinib, OSI-930, pazopanib (GW786034), pexidartinib (PLX3397),PKC412, PLX647, PP1, quizartinib (AC220), regorafenib (BAY 73-4506),semaxinib (SU 5416), sitravatinib (MGCD516), sorafenib, STI571, SU11248,SU9529, sunitinib, telatinib, tivozanib (AV-951), tyrphostin AG 1296,VX-322, and WBZ 4.

Exemplary MDM2 inhibitors include: (−)-parthenolide, ALRN6924, AM-8553,AMG232, CGM-097, DS-3032b, GEM240, HDM201, H1198, idasanutlin (RG-7338),JapA, MI-219, MI-219, MI-319, MI-77301 (SAR405838), MK4828, MK-8242,MX69, NSC 207895 (XI-006), Nutlin-3, Nutlin-3a, Nutlin-3b, NVP-CFC218,NVP-CGM097, PXn727/822, RG7112, RO2468, RO5353, RO5503781, serdemetan(JNJ-26854165), SP-141, and YH239-EE.

Exemplary inhibitors of amplified MDM2 include: AM-8553, AMG232,DS-3032b, MI-77301 (SAR405838), NSC 207895 (XI-006), Nutlin-3a,NVP-CFC218, NVP-CGM097, and RG7112.

Exemplary inhibitors of MET include: (−)-Oleocanthal, ABBV-399, AMG-208,AMG-337, AMG-458, BAY-853474, BMS-754807, BMS-777607, BMS-794833,cabozantinib (XL184, BMS-907351), capmatinib (INCB28060), crizotinib(PF-02341066), DE605, foretinib (GSK1363089, XL880), glesatinib(MGCD265), golvatinib (E7050), INCB028060, JNJ-38877605, KRC-408,merestinib (LY2801653), MK-2461, MK8033, NPS-1034, NVP-BVU972,PF-04217903, PHA-665752, 549076, savolitinib (AZD6094, HMPL-504),SGX-523, SU11274, TAS-115, tepotinib (EMD 1214063, MSC2156119J),volitinib, CE-355621, and Onartuzumab.

Exemplary inhibitors of mTOR include: anthracimycin, apitolisib(GDC-0980, RG7422), AZD-8055, BGT226 (NVP-BGT226), CC-223, CZ415,dactolisib (BEZ235, NVP-BEZ235), DS7423, everolimus (RAD001), GDC-0084,GDC-0349, gedatolisib (PF-05212384, PKI-5587), GSK1059615, INK128,KU-0063794, LY3023414, MLN0128, omipalisib (GSK2126458, GSK458),OSI-027, OSU-53, Palomid 529 (P529), PF-04691502, PI-103, PKI-587,PP242, PQR309, ridafarolimus (AP-23573), sapanisertib (INK 128,MLN0128), SAR245409 (XL765), SF-1126, SF2523, sirolimus (rapamycin),SN32976, TAK228, temsirolimus (CCI-779, NSC 683864), Torin 1, Torin 2,torkinib (PP242), umirolimus, vistusertib (AZD2014), voxtalisib (XL765,SAR245409), VS-5584, VS-5584 (SB2343), WAY-600, WYE-125132 (WYE-132),WYE-354, WYE-687, XL388, and zotarolimus (ABT-578).

Exemplary inhibitors of MYC include: 10058-F4, 10074-G5, and KSI-3716.

The phrase “dysregulation of a gene, a protein, or the expression oractivity or level of any of the same” refers to a genetic mutation(e.g., a chromosomal translocation that results in the expression of afusion protein including a kinase domain and a fusion partner, amutation in a gene that results in the expression of a protein thatincludes a deletion of at least one amino acid as compared to a wildtypeprotein, a mutation in a gene that results in the expression of aprotein with one or more point mutations as compared to a wildtypeprotein, a mutation in a gene that results in the expression of aprotein with at least one inserted amino acid as compared to a wildtypeprotein, a gene duplication that results in an increased level ofprotein in a cell, or a mutation in a regulatory sequence (e.g., apromoter and/or enhancer) that results in an increased level of proteinin a cell), an alternative spliced version of a mRNA that results in aprotein having a deletion of at least one amino acid in the protein ascompared to the wild-type protein), or increased expression (e.g.,increased levels) of a wildtype protein in a mammalian cell due toaberrant cell signaling and/or dysregulated autocrine/paracrinesignaling (e.g., as compared to a control non-cancerous cell). Asanother example, a dysregulation of a gene, a protein, or expression oractivity, or level of any of the same, can be a mutation in a gene thatencodes a protein that is constitutively active or has increasedactivity as compared to a protein encoded by a gene that does notinclude the mutation. For example, a dysregulation of a gene, a protein,or expression or activity, or level of any of the same, can be theresult of a gene or chromosome translocation which results in theexpression of a fusion protein that contains a first portion of aprotein that includes a functional kinase domain, and a second portionof a partner protein (i.e., that is not the primary protein). In someexamples, dysregulation of a gene, a protein, or expression or activityor level of any of the same can be a result of a gene translocation ofone gene with a different gene.

Treatment of a patient having a cancer with a multi-kinase inhibitor(MKI) or target-specific kinase inhibitor (e.g., a BRAF inhibitor, aEGFR inhibitor, a MEK inhibitor, an ALK inhibitor, a ROS1 inhibitor, aMET inhibitor, an aromatase inhibitor, a RAF inhibitor, or a RASinhibitor) can result in dysregulation of a RET gene, a RET kinase, orthe expression or activity or level of the same in the cancer, and/orresistance to a RET inhibitor. See, e.g., Bhinge et al., Oncotarget8:27155-27165, 2017; Chang et al., Yonsei Med. J. 58:9-18, 2017; andLopez-Delisle et al., doi: 10.1038/s41388-017-0039-5, Oncogene 2018.

Treatment of a patient having a cancer with a RET inhibitor incombination with a multi-kinase inhibitor or a target-specific kinaseinhibitor (e.g., a BRAF inhibitor, a EGFR inhibitor, a MEK inhibitor, anALK inhibitor, a ROS1 inhibitor, a MET inhibitor, an aromataseinhibitor, a RAF inhibitor, or a RAS inhibitor) can have increasedtherapeutic efficacy as compared to treatment of the same patient or asimilar patient with the RET inhibitor as a monotherapy, or themulti-kinase inhibitor or the target-specific kinase inhibitor as amonotherapy. See, e.g., Tang et al., doi: 10.1038/modpathol.2017.109,Mod. Pathol. 2017; Andreucci et al., Oncotarget 7:80543-80553, 2017;Nelson-Taylor et al., Mol. Cancer Ther. 16:1623-1633, 2017; and Kato etal., Clin. Cancer Res. 23:1988-1997, 2017.

Provided herein are methods of treating a patient having a cancer (e.g.,any of the cancers described herein) and previously administered amulti-kinase inhibitor (MKI) or a target-specific kinase inhibitor(e.g., a BRAF inhibitor, a EGFR inhibitor, a MEK inhibitor, an ALKinhibitor, a ROS1 inhibitor, a MET inhibitor, an aromatase inhibitor, aRAF inhibitor, or a RAS inhibitor) (e.g., as a monotherapy) thatinclude: administering to the patient (i) a therapeutically effectivedose of a compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof as a monotherapy, or (ii) atherapeutically effective dose of a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,and a therapeutically effective dose of the previously administered MKIor the previously administered target-specific kinase inhibitor.

Provided herein are methods of treating a patient having a cancer (e.g.,any of the cancers described herein) previously administered a MKI or atarget specific kinase inhibitor (e.g., a BRAF inhibitor, a EGFRinhibitor, a MEK inhibitor, an ALK inhibitor, a ROS1 inhibitor, a METinhibitor, an aromatase inhibitor, a RAF inhibitor, or a RAS inhibitor)(e.g., as a monotherapy) that include: identifying a patient having acancer cell that has a dysregulation of a RET gene, a RET kinase, or theexpression or activity or level of the same; and administering to theidentified patient (i) a therapeutically effective dose of a compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof as a monotherapy, or (ii) a therapeuticallyeffective dose of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof, and atherapeutically effective dose of the previously administered MKI or thepreviously administered target-specific kinase inhibitor.

Provided herein are methods of treating a patient having a cancer (e.g.,any of the cancers described herein) that include: administering to apatient a therapeutically effective amount of a MKI or a target-specifickinase inhibitor (e.g., a BRAF inhibitor, a EGFR inhibitor, a MEKinhibitor, an ALK inhibitor, a ROS1 inhibitor, a MET inhibitor, anaromatase inhibitor, a RAF inhibitor, or a RAS inhibitor) (e.g., as amonotherapy) for a first period of time; after the period of time,identifying a patient having a cancer cell that has a dysregulation of aRET gene, a RET kinase, or the expression or activity or level of thesame; and administering to the identified patient (i) a therapeuticallyeffective dose of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof as a monotherapy,or (ii) a therapeutically effective dose of a compound of Formula I-IV,or a pharmaceutically acceptable salt, amorphous, or polymorph formthereof, and a therapeutically effective dose of the previouslyadministered MKI or the previously administered target-specific kinaseinhibitor.

Provided herein are methods of treating a patient having a cancer (e.g.,any of the cancers described herein) that has dysregulation of a BRAFgene, a BRAF kinase, or the expression or activity or level of the samethat include administering to the patient (i) a therapeuticallyeffective amount of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof and (ii) atherapeutically effective amount of a BRAF inhibitor (e.g., any of theBRAF inhibitors described herein or known in the art).

Provided herein are methods of treating a patient having a cancer (e.g.,any of the cancers described herein) that include: identifying a patienthaving a cancer cell that has dysregulation of a BRAF gene, a BRAFkinase, or the expression or activity or level of the same; andadministering to the identified patient (i) a therapeutically effectiveamount of a compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof and (ii) a therapeuticallyeffective amount of a BRAF inhibitor (e.g., any of the BRAF inhibitorsdescribed herein or known in the art).

Provided herein are methods of treating a patient having a cancer (e.g.,any of the cancers described herein) that has dysregulation of an EGFRgene, an EGFR protein, or the expression or activity or level of thesame that include administering to the patient (i) a therapeuticallyeffective amount of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof and (ii) atherapeutically effective amount of an EGFR inhibitor (e.g., any of theEGFR inhibitors described herein or known in the art).

Provided herein are methods of treating a patient having a cancer (e.g.,any of the cancers described herein) that include: identifying a patienthaving a cancer cell that has dysregulation of an EGFR gene, an EGFRprotein, or the expression or activity or level of the same; andadministering to the identified patient (i) a therapeutically effectiveamount of a compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof and (ii) a therapeuticallyeffective amount of an EGFR inhibitor (e.g., any of the EGFR inhibitorsdescribed herein or known in the art).

Provided herein are methods of treating a patient having a cancer (e.g.,any of the cancers described herein) that has dysregulation of a MEKgene, a MEK protein, or the expression or activity or level of the samethat include administering to the patient (i) a therapeuticallyeffective amount of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof and (ii) atherapeutically effective amount of a MEK inhibitor (e.g., any of theMEK inhibitors described herein or known in the art).

Provided herein are methods of treating a patient having a cancer (e.g.,any of the cancers described herein) that include: identifying a patienthaving a cancer cell that has dysregulation of a MEK gene, a MEKprotein, or the expression or activity or level of the same; andadministering to the identified patient (i) a therapeutically effectiveamount of a compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof and (ii) a therapeuticallyeffective amount of a MEK inhibitor (e.g., any of the MEK inhibitorsdescribed herein or known in the art).

Provided herein are methods of treating a patient having a cancer (e.g.,any of the cancers described herein) that has dysregulation of an ALKgene, an ALK protein, or the expression or activity or level of the samethat include administering to the patient (i) a therapeuticallyeffective amount of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof and (ii) atherapeutically effective amount of an ALK inhibitor (e.g., any of theALK inhibitors described herein or known in the art).

Provided herein are methods of treating a patient having a cancer (e.g.,any of the cancers described herein) that include: identifying a patienthaving a cancer cell that has dysregulation of an ALK gene, an ALKprotein, or the expression or activity or level of the same; andadministering to the identified patient (i) a therapeutically effectiveamount of a compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof and (ii) a therapeuticallyeffective amount an ALK inhibitor (e.g., any of the ALK inhibitorsdescribed herein or known in the art).

Provided herein are methods of treating a patient having a cancer (e.g.,any of the cancers described herein) that has dysregulation of a ROSgene, a ROS protein, or the expression or activity or level of the samethat include administering to the patient (i) a therapeuticallyeffective amount of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof and (ii) atherapeutically effective amount of a ROS inhibitor (e.g., any of theROS inhibitors described herein or known in the art).

Provided herein are methods of treating a patient having a cancer (e.g.,any of the cancers described herein) that include: identifying a patienthaving a cancer cell that has dysregulation of a ROS gene, a ROSprotein, or the expression or activity or level of the same; andadministering to the identified patient (i) a therapeutically effectiveamount of a compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof and (ii) a therapeuticallyeffective amount a ROS inhibitor (e.g., any of the ROS inhibitorsdescribed herein or known in the art).

Provided herein are methods of treating a patient having a cancer (e.g.,any of the cancers described herein) that has dysregulation of a METgene, a MET protein, or the expression or activity or level of the samethat include administering to the patient (i) a therapeuticallyeffective amount of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof and (ii) atherapeutically effective amount of a MET inhibitor (e.g., any of theMET inhibitors described herein or known in the art).

Provided herein are methods of treating a patient having a cancer (e.g.,any of the cancers described herein) that include: identifying a patienthaving a cancer cell that has dysregulation of a MET gene, a METprotein, or the expression or activity or level of the same; andadministering to the identified patient (i) a therapeutically effectiveamount of a compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof and (ii) a therapeuticallyeffective amount a MET inhibitor (e.g., any of the MET inhibitorsdescribed herein or known in the art).

Provided herein are methods of treating a patient having a cancer (e.g.,any of the cancers described herein) that has dysregulation of anaromatase gene, an aromatase protein, or the expression or activity orlevel of the same that include administering to the patient (i) atherapeutically effective amount of a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofand (ii) a therapeutically effective amount of an aromatase inhibitor(e.g., any of the aromatase inhibitors described herein or known in theart).

Provided herein are methods of treating a patient having a cancer (e.g.,any of the cancers described herein) that include: identifying a patienthaving a cancer cell that has dysregulation of an aromatase gene, anaromatase protein, or the expression or activity or level of the same;and administering to the identified patient (i) a therapeuticallyeffective amount of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof and (ii) atherapeutically effective amount an aromatase inhibitor (e.g., any ofthe aromatase inhibitors described herein or known in the art).

Provided herein are methods of treating a patient having a cancer (e.g.,any of the cancers described herein) that has dysregulation of a RAFgene, a RAF protein, or the expression or activity or level of the samethat include administering to the patient (i) a therapeuticallyeffective amount of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof and (ii) atherapeutically effective amount of a RAF inhibitor (e.g., any of theRAF inhibitors described herein or known in the art).

Provided herein are methods of treating a patient having a cancer (e.g.,any of the cancers described herein) that include: identifying a patienthaving a cancer cell that has dysregulation of a RAF gene, a RAFprotein, or the expression or activity or level of the same; andadministering to the identified patient (i) a therapeutically effectiveamount of a compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof and (ii) a therapeuticallyeffective amount a RAF inhibitor (e.g., any of the RAF inhibitorsdescribed herein or known in the art).

Provided herein are methods of treating a patient having a cancer (e.g.,any of the cancers described herein) that has dysregulation of a RASgene, a RAS protein, or the expression or activity or level of the samethat include administering to the patient (i) a therapeuticallyeffective amount of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof and (ii) atherapeutically effective amount of a RAS inhibitor (e.g., any of theRAS inhibitors described herein or known in the art).

Provided herein are methods of treating a patient having a cancer (e.g.,any of the cancers described herein) that include: identifying a patienthaving a cancer cell that has dysregulation of a RAS gene, a RASprotein, or the expression or activity or level of the same; andadministering to the identified patient (i) a therapeutically effectiveamount of a compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof and (ii) a therapeuticallyeffective amount a RAS inhibitor (e.g., any of the RAS inhibitorsdescribed herein or known in the art).

The phrase “dysregulation of a BRAF gene, a BRAF protein, or theexpression or activity or level of any of the same” refers to a geneticmutation (e.g., a chromosomal translocation that results in theexpression of a fusion protein including a BRAF kinase domain and afusion partner, a mutation in a BRAF gene that results in the expressionof a BRAF protein that includes a deletion of at least one amino acid ascompared to a wildtype BRAF protein, a mutation in a BRAF gene thatresults in the expression of a BRAF protein with one or more pointmutations as compared to a wildtype BRAF protein, a mutation in a BRAFgene that results in the expression of a BRAF protein with at least oneinserted amino acid as compared to a wildtype BRAF protein, a geneduplication that results in an increased level of BRAF protein in acell, or a mutation in a regulatory sequence (e.g., a promoter and/orenhancer) that results in an increased level of BRAF protein in a cell),an alternative spliced version of a BRAF mRNA that results in a BRAFprotein having a deletion of at least one amino acid in the BRAF proteinas compared to the wild-type BRAF protein), or increased expression(e.g., increased levels) of a wildtype BRAF protein in a mammalian celldue to aberrant cell signaling and/or dysregulated autocrine/paracrinesignaling (e.g., as compared to a control non-cancerous cell). Asanother example, a dysregulation of a BRAF gene, a BRAF protein, orexpression or activity, or level of any of the same, can be a mutationin a BRAF gene that encodes a BRAF protein that is constitutively activeor has increased activity as compared to a protein encoded by a BRAFgene that does not include the mutation. For example, a dysregulation ofa BRAF gene, a BRAF protein, or expression or activity, or level of anyof the same, can be the result of a gene or chromosome translocationwhich results in the expression of a fusion protein that contains afirst portion of a BRAF protein that includes a functional kinasedomain, and a second portion of a partner protein (i.e., that is notBRAF). In some examples, dysregulation of a BRAF gene, a BRAF protein,or expression or activity or level of any of the same can be a result ofa gene translocation of one BRAF gene with another non-BRAF gene.

Non-limiting examples of a BRAF inhibitor include dabrafenib,vemurafenib (also called RG7204 or PLX4032), sorafenib tosylate,PLX-4720, GDC-0879, BMS-908662 (Bristol-Meyers Squibb), LGX818(Novartis), PLX3603 (Hofmann-LaRoche), RAF265 (Novartis), RO5185426(Hofmann-LaRoche), and GSK2118436 (GlaxoSmithKline). Additional examplesof a BRAF inhibitor are known in the art.

The phrase “dysregulation of an EGFR gene, an EGFR protein, or theexpression or activity or level of any of the same” refers to a geneticmutation (e.g., a chromosomal translocation that results in theexpression of a fusion protein including an EGFR kinase domain and afusion partner, a mutation in an EGFR gene that results in theexpression of an EGFR protein that includes a deletion of at least oneamino acid as compared to a wildtype EGFR protein, a mutation in an EGFRgene that results in the expression of an EGFR protein with one or morepoint mutations as compared to a wildtype EGFR protein, a mutation in anEGFR gene that results in the expression of an EGFR protein with atleast one inserted amino acid as compared to a wildtype EGFR protein, agene duplication that results in an increased level of EGFR protein in acell, or a mutation in a regulatory sequence (e.g., a promoter and/orenhancer) that results in an increased level of EGFR protein in a cell),an alternative spliced version of a EGFR mRNA that results in an EGFRprotein having a deletion of at least one amino acid in the EGFR proteinas compared to the wild-type EGFR protein), or increased expression(e.g., increased levels) of a wildtype EGFR protein in a mammalian celldue to aberrant cell signaling and/or dysregulated autocrine/paracrinesignaling (e.g., as compared to a control non-cancerous cell). Asanother example, a dysregulation of an EGFR gene, an EGFR protein, orexpression or activity, or level of any of the same, can be a mutationin an EGFR gene that encodes an EGFR protein that is constitutivelyactive or has increased activity as compared to a protein encoded by anEGFR gene that does not include the mutation. For example, adysregulation of an EGFR gene, an EGFR protein, or expression oractivity, or level of any of the same, can be the result of a gene orchromosome translocation which results in the expression of a fusionprotein that contains a first portion of a EGFR protein that includes afunctional kinase domain, and a second portion of a partner protein(i.e., that is not EGFR). In some examples, dysregulation of an EGFRgene, an EGFR protein, or expression or activity or level of any of thesame can be a result of a gene translocation of one EGFR gene withanother non-EGFR gene.

Non-limiting examples of an EGFR inhibitor include gefitinib, erlotinib,brigatinib, lapatinib, neratinib, icotinib, afatinib, dacomitinib,poziotinib, vandetanib, afatinib, AZD9291, CO-1686, HM61713, AP26113,CI-1033, PKI-166, GW-2016, EKB-569, PDI-168393, AG-1478, CGP-59326A.Additional examples of an EGFR inhibitor are known in the art.

The phrase “dysregulation of a MEK gene, a MEK protein, or theexpression or activity or level of any of the same” refers to a geneticmutation (e.g., a chromosomal translocation that results in theexpression of a fusion protein including a MEK kinase domain and afusion partner, a mutation in a MEK gene that results in the expressionof a MEK protein that includes a deletion of at least one amino acid ascompared to a wildtype MEK protein, a mutation in a MEK gene thatresults in the expression of a MEK protein with one or more pointmutations as compared to a wildtype MEK protein, a mutation in a MEKgene that results in the expression of a MEK protein with at least oneinserted amino acid as compared to a wildtype MEK protein, a geneduplication that results in an increased level of MEK protein in a cell,or a mutation in a regulatory sequence (e.g., a promoter and/orenhancer) that results in an increased level of MEK protein in a cell),an alternative spliced version of a MEK mRNA that results in a MEKprotein having a deletion of at least one amino acid in the MEK proteinas compared to the wild-type MEK protein), or increased expression(e.g., increased levels) of a wildtype MEK protein in a mammalian celldue to aberrant cell signaling and/or dysregulated autocrine/paracrinesignaling (e.g., as compared to a control non-cancerous cell). Asanother example, a dysregulation of a MEK gene, a MEK protein, orexpression or activity, or level of any of the same, can be a mutationin a MEK gene that encodes a MEK protein that is constitutively activeor has increased activity as compared to a protein encoded by a MEK genethat does not include the mutation. For example, a dysregulation of aMEK gene, a MEK protein, or expression or activity, or level of any ofthe same, can be the result of a gene or chromosome translocation whichresults in the expression of a fusion protein that contains a firstportion of a MEK protein that includes a functional kinase domain, and asecond portion of a partner protein (i.e., that is not MEK). In someexamples, dysregulation of a MEK gene, a MEK protein, or expression oractivity or level of any of the same can be a result of a genetranslocation of one MEK gene with another non-MEK gene.

Non-limiting examples of a MEK inhibitor include mekinist, trametinib(GSK1120212), cobimetinib (XL518), binimetinib (MEK162), selumetinib,PD-325901, CI-1040, PD035901, TAK-733, PD098059, U0126,AS703026/MSC1935369, XL-518/GDC-0973, BAY869766/RDEA119, and GSK1120212.Additional examples of a MEK inhibitor are known in the art.

The phrase “dysregulation of an ALK gene, an ALK protein, or theexpression or activity or level of any of the same” refers to a geneticmutation (e.g., a chromosomal translocation that results in theexpression of a fusion protein including an ALK kinase domain and afusion partner, a mutation in an ALK gene that results in the expressionan ALK protein that includes a deletion of at least one amino acid ascompared to a wildtype ALK protein, a mutation in an ALK gene thatresults in the expression of an ALK protein with one or more pointmutations as compared to a wildtype ALK protein, a mutation in an ALKgene that results in the expression of an ALK protein with at least oneinserted amino acid as compared to a wildtype ALK protein, a geneduplication that results in an increased level of ALK protein in a cell,or a mutation in a regulatory sequence (e.g., a promoter and/orenhancer) that results in an increased level of ALK protein in a cell),an alternative spliced version of an ALK mRNA that results in an ALKprotein having a deletion of at least one amino acid in the ALK proteinas compared to the wild-type ALK protein), or increased expression(e.g., increased levels) of a wildtype ALK protein in a mammalian celldue to aberrant cell signaling and/or dysregulated autocrine/paracrinesignaling (e.g., as compared to a control non-cancerous cell). Asanother example, a dysregulation of an ALK gene, an ALK protein, orexpression or activity, or level of any of the same, can be a mutationin an ALK gene that encodes an ALK protein that is constitutively activeor has increased activity as compared to a protein encoded by an ALKgene that does not include the mutation. For example, a dysregulation ofan ALK gene, an ALK protein, or expression or activity, or level of anyof the same, can be the result of a gene or chromosome translocationwhich results in the expression of a fusion protein that contains afirst portion of an ALK protein that includes a functional kinasedomain, and a second portion of a partner protein (i.e., that is notALK). In some examples, dysregulation of an ALK gene, an ALK protein, orexpression or activity or level of any of the same can be a result of agene translocation of one ALK gene with another non-ALK gene.

Non-limiting examples of an ALK inhibitor include crizotinib (Xalkori),ceritinib (Zykadia), alectinib (Alecensa), dalantercept, ACE-041(Brigatinib) (AP26113), entrectinib (NMS-E628), PF-06463922 (Pfizer),TSR-011 (Tesaro), CEP-37440 (Teva), CEP-37440 (Teva), X-396 (Xcovery),and ASP-3026 (Astellas). Additional examples of an ALK inhibitor areknown in the art.

The phrase “dysregulation of a ROS1 gene, a ROS1 protein, or theexpression or activity or level of any of the same” refers to a geneticmutation (e.g., a chromosomal translocation that results in theexpression of a fusion protein including a ROS1 kinase domain and afusion partner, a mutation in a ROS1 gene that results in the expressiona ROS1 protein that includes a deletion of at least one amino acid ascompared to a wildtype ROS1 protein, a mutation in a ROS1 gene thatresults in the expression of a ROS1 protein with one or more pointmutations as compared to a wildtype ROS1 protein, a mutation in a ROS1gene that results in the expression of a ROS1 protein with at least oneinserted amino acid as compared to a wildtype ROS1 protein, a geneduplication that results in an increased level of ROS1 protein in acell, or a mutation in a regulatory sequence (e.g., a promoter and/orenhancer) that results in an increased level of ROS1 protein in a cell),an alternative spliced version of a ROS1 mRNA that results in a ROS1protein having a deletion of at least one amino acid in the ROS1 proteinas compared to the wild-type ROS1 protein), or increased expression(e.g., increased levels) of a wildtype ROS1 protein in a mammalian celldue to aberrant cell signaling and/or dysregulated autocrine/paracrinesignaling (e.g., as compared to a control non-cancerous cell). Asanother example, a dysregulation of a ROS1 gene, a ROS1 protein, orexpression or activity, or level of any of the same, can be a mutationin a ROS1 gene that encodes a ROS1 protein that is constitutively activeor has increased activity as compared to a protein encoded by a ROS1gene that does not include the mutation. For example, a dysregulation ofa ROS1 gene, a ROS1 protein, or expression or activity, or level of anyof the same, can be the result of a gene or chromosome translocationwhich results in the expression of a fusion protein that contains afirst portion of a ROS1 protein that includes a functional kinasedomain, and a second portion of a partner protein (i.e., that is notROS1). In some examples, dysregulation of a ROS1 gene, a ROS1 protein,or expression or activity or level of any of the same can be a result ofa gene translocation of one ROS1 gene with another non-ROS1 gene.

Non-limiting examples of a ROS1 inhibitor include crizotinib,entrectinib (RXDX-101), lorlatinib (PF-06463922), certinib, TPX-0005,DS-605, and cabozantinib. Additional examples of a ROS1 inhibitor areknown in the art.

The phrase “dysregulation of a MET gene, a MET protein, or theexpression or activity or level of any of the same” refers to a geneticmutation (e.g., a chromosomal translocation that results in theexpression of a fusion protein including a MET kinase domain and afusion partner, a mutation in a MET gene that results in the expressiona MET protein that includes a deletion of at least one amino acid ascompared to a wildtype MET protein, a mutation in a MET gene thatresults in the expression of a MET protein with one or more pointmutations as compared to a wildtype MET protein, a mutation in a METgene that results in the expression of a MET protein with at least oneinserted amino acid as compared to a wildtype MET protein, a geneduplication that results in an increased level of MET protein in a cell,or a mutation in a regulatory sequence (e.g., a promoter and/orenhancer) that results in an increased level of MET protein in a cell),an alternative spliced version of a MET mRNA that results in a METprotein having a deletion of at least one amino acid in the MET proteinas compared to the wild-type MET protein), or increased expression(e.g., increased levels) of a wildtype MET protein in a mammalian celldue to aberrant cell signaling and/or dysregulated autocrine/paracrinesignaling (e.g., as compared to a control non-cancerous cell). Asanother example, a dysregulation of a MET gene, a MET protein, orexpression or activity, or level of any of the same, can be a mutationin a MET gene that encodes a MET protein that is constitutively activeor has increased activity as compared to a protein encoded by a MET genethat does not include the mutation. For example, a dysregulation of aMET gene, a MET protein, or expression or activity, or level of any ofthe same, can be the result of a gene or chromosome translocation whichresults in the expression of a fusion protein that contains a firstportion of a MET protein that includes a functional kinase domain, and asecond portion of a partner protein (i.e., that is not MET). In someexamples, dysregulation of a MET gene, a MET protein, or expression oractivity or level of any of the same can be a result of a genetranslocation of one MET gene with another non-MET gene.

Non-limiting examples of a MET inhibitor include crizotinib,cabozantinib, JNJ-38877605, PF-04217903 (Pfizer), MK-2461, GSK 1363089,AMG 458 (Amgen), tivantinib, INCB28060 (Incyte), PF-02341066 (Pfizer),E7050 (Eisai), BMS-777607 (Bristol-Meyers Squibb), JNJ-38877605 (Johnson& Johnson), ARQ197 (ArQule), GSK/1363089/XL880 (GSK/Exeilixis), andXL174 (BMS/Exelixis). Additional examples of a MET inhibitor are knownin the art.

The phrase “dysregulation of a aromatase gene, an aromatase protein, orthe expression or activity or level of any of the same” refers to agenetic mutation (e.g., a mutation in an aromatase gene that results inthe expression an aromatase protein that includes a deletion of at leastone amino acid as compared to a wildtype aromatase protein, a mutationin an aromatase gene that results in the expression of an aromataseprotein with one or more point mutations as compared to a wildtypearomatase protein, a mutation in an aromatase gene that results in theexpression of an aromatase protein with at least one inserted amino acidas compared to a wildtype aromatase protein, a gene duplication thatresults in an increased level of aromatase protein in a cell, or amutation in a regulatory sequence (e.g., a promoter and/or enhancer)that results in an increased level of aromatase protein in a cell), analternative spliced version of an aromatase mRNA that results in anaromatase protein having a deletion of at least one amino acid in thearomatase protein as compared to the wild-type aromatase protein), orincreased expression (e.g., increased levels) of a wildtype aromatase ina mammalian cell due to aberrant cell signaling and/or dysregulatedautocrine/paracrine signaling (e.g., as compared to a controlnon-cancerous cell). As another example, a dysregulation of an aromatasegene, an aromatase protein, or expression or activity, or level of anyof the same, can be a mutation in an aromatase gene that encodes anaromatase protein that is constitutively active or has increasedactivity as compared to a protein encoded by an aromatase gene that doesnot include the mutation.

Non-limiting examples of an aromatase inhibitor include Arimidex(anastrozole), Aromasin (exemestane), Femara (letrozole), Teslac(testolactone), and formestane. Additional examples of an aromataseinhibitor are known in the art.

The phrase “dysregulation of a RAF gene, a RAF protein, or theexpression or activity or level of any of the same” refers to a geneticmutation (e.g., a chromosomal translocation that results in theexpression of a fusion protein including a RAF kinase domain and afusion partner, a mutation in a RAF gene that results in the expressiona RAF protein that includes a deletion of at least one amino acid ascompared to a wildtype RAF protein, a mutation in a RAF gene thatresults in the expression of a RAF protein with one or more pointmutations as compared to a wildtype RAF protein, a mutation in a RAFgene that results in the expression of a RAF protein with at least oneinserted amino acid as compared to a wildtype RAF protein, a geneduplication that results in an increased level of RAF protein in a cell,or a mutation in a regulatory sequence (e.g., a promoter and/orenhancer) that results in an increased level of RAF protein in a cell),an alternative spliced version of a RAF mRNA that results in a RAFprotein having a deletion of at least one amino acid in the RAF proteinas compared to the wild-type RAF protein), or increased expression(e.g., increased levels) of a wildtype RAF protein in a mammalian celldue to aberrant cell signaling and/or dysregulated autocrine/paracrinesignaling (e.g., as compared to a control non-cancerous cell). Asanother example, a dysregulation of a RAF gene, a RAF protein, orexpression or activity, or level of any of the same, can be a mutationin a RAF gene that encodes a RAF protein that is constitutively activeor has increased activity as compared to a protein encoded by a RAF genethat does not include the mutation. For example, a dysregulation of aRAF gene, a RAF protein, or expression or activity, or level of any ofthe same, can be the result of a gene or chromosome translocation whichresults in the expression of a fusion protein that contains a firstportion of a RAF protein that includes a functional kinase domain, and asecond portion of a partner protein (i.e., that is not RAF). In someexamples, dysregulation of a RAF gene, a RAF protein, or expression oractivity or level of any of the same can be a result of a genetranslocation of one RAF gene with another non-RAF gene.

Non-limiting examples of a RAF inhibitor include sorafenib, vemurafenib,dabrafenib, BMS-908662/XL281, GSK2118436, RAF265, RO5126766, andRO4987655. Additional examples of a RAF inhibitor are known in the art.

The phrase “dysregulation of a RAS gene, a RAS protein, or theexpression or activity or level of any of the same” refers to a geneticmutation (e.g., a chromosomal translocation that results in theexpression of a fusion protein including a RAS kinase domain and afusion partner, a mutation in a RAS gene that results in the expressiona RAS protein that includes a deletion of at least one amino acid ascompared to a wildtype RAS protein, a mutation in a RAS gene thatresults in the expression of a RAS protein with one or more pointmutations as compared to a wildtype RAS protein, a mutation in a RASgene that results in the expression of a RAS protein with at least oneinserted amino acid as compared to a wildtype RAS protein, a geneduplication that results in an increased level of RAS protein in a cell,or a mutation in a regulatory sequence (e.g., a promoter and/orenhancer) that results in an increased level of RAS protein in a cell),an alternative spliced version of a RAS mRNA that results in a RASprotein having a deletion of at least one amino acid in the RAS proteinas compared to the wild-type RAS protein), or increased expression(e.g., increased levels) of a wildtype RAS protein in a mammalian celldue to aberrant cell signaling and/or dysregulated autocrine/paracrinesignaling (e.g., as compared to a control non-cancerous cell). Asanother example, a dysregulation of a RAS gene, a RAS protein, orexpression or activity, or level of any of the same, can be a mutationin a RAS gene that encodes a RAS protein that is constitutively activeor has increased activity as compared to a protein encoded by a RAS genethat does not include the mutation. For example, a dysregulation of aRAS gene, a RAS protein, or expression or activity, or level of any ofthe same, can be the result of a gene or chromosome translocation whichresults in the expression of a fusion protein that contains a firstportion of a RAS protein that includes a functional kinase domain, and asecond portion of a partner protein (i.e., that is not RAS). In someexamples, dysregulation of a RAS gene, a RAS protein, or expression oractivity or level of any of the same can be a result of a genetranslocation of one RAS gene with another non-RAS gene.

Non-limiting examples of a RAS inhibitor include Kobe0065 and Kobe2602.Additional examples of a RAS inhibitor are known in the art.

Non-limiting examples of multi-kinase inhibitors (MKIs) includedasatinib and sunitinib.

In some embodiments, provided herein are methods of treating a subjecthaving a cancer that include: (a) administering one or more doses of afirst RET inhibitor or a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof to the subject fora period of time; (b) after (a), determining whether a cancer cell in asample obtained from the subject has at least one dysregulation of agene, a protein, or the expression or activity or level of any of thesame, wherein the gene or protein is selected from the group consistingof EGFR, MET, ALK, ROS1, KRAS, BRAF, RAS, PIK3CA, and HER2; and (c)administering 1) a second RET inhibitor as a monotherapy or inconjunction with another anticancer agent, 2) administering additionaldoses of the first RET inhibitor or a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofin combination with an inhibitor targeting the gene or protein (e.g., aninhibitor of EGFR, MET, ALK, ROS1, KRAS, BRAF, RAS, PIK3CA, and HER2),or 3) stop administration of the RET inhibitor of step a) and administeran inhibitor targeting the gene or protein (e.g., an inhibitor of EGFR,MET, ALK, ROS1, KRAS, BRAF, RAS, PIK3CA, and HER2) to the subject if thesubject has a cancer cell that has at least one dysregulation of a gene,a protein, or the expression or activity or level of the same, whereinthe gene or protein is selected from the group consisting of EGFR, MET,ALK, ROS1, KRAS, BRAF, RAS, PIK3CA, and HER2; or (d) administeringadditional doses of the first RET inhibitor step (a) to the subject ifthe subject has a cancer cell that does not have a RET inhibitorresistance mutation. In some embodiments, the one or more dysregulationsof a gene, a protein, or the expression or activity or level of any ofthe same, wherein the gene or protein is selected from the groupconsisting of EGFR, MET, ALK, ROS1, KRAS, BRAF, RAS, PIK3CA, and HER2confer increased resistance to a cancer cell or tumor to treatment withthe first RET inhibitor or the compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof.In some embodiments, the tumor is a NSCLC tumor and the one or moredysregulations of a gene, a protein, or the expression or activity orlevel of any of the same are selected from targetable mutations in EGFRor MET, targetable rearrangements involving ALK or ROS1, or activatingmutations in KRAS. In some embodiments, the tumor is a thyroid (non-MTC)tumor and the one or more dysregulations of a gene, a protein, or theexpression or activity or level of any of the same are selected fromtargetable mutations in BRAF or activating mutations in RAS. In someembodiments, the tumor is a MTC tumor and the one or more dysregulationsof a gene, a protein, or the expression or activity or level of any ofthe same are selected from targetable mutations in ALK or activatingmutations in RAS. In some embodiments, the tumor is a pancreatic tumorand the one or more dysregulations of a gene, a protein, or theexpression or activity or level of any of the same is an activatingmutations in KRAS. In some embodiments, the tumor is a colorectal tumorand the one or more dysregulations of a gene, a protein, or theexpression or activity or level of any of the same are selected fromtargetable mutations in BRAF or PIK3CA or an activating mutation in RAS.In some embodiments, the tumor is a breast tumor and the one or moredysregulations of a gene, a protein, or the expression or activity orlevel of any of the same are selected from targetable mutations inPIK3CA or alteration in HER2.

Also provided are methods of selecting a treatment for a subject havinga cancer that include (a) administering one or more doses of a first RETinhibitor to the subject for a period of time; (b) after (a),determining whether a cancer cell in a sample obtained from the subjecthas at least one RET inhibitor resistance mutation; and (c) selecting acompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof as a monotherapy or in conjunctionwith another anticancer agent for the subject if the subject has acancer cell that has one or more RET inhibitor resistance mutations; or(d) selecting additional doses of the first RET inhibitor of step (a)for the subject if the subject has a cancer cell that does not have aRET inhibitor resistance mutation. In some embodiments, when additionaldoses of the first RET inhibitor of step (a) are selected for thesubject, the method can further include selecting doses of anotheranticancer agent for the subject. In some embodiments, the one or moreRET inhibitor resistance mutations confer increased resistance to acancer cell or tumor to treatment with the first RET inhibitor. In someembodiments, the one or more RET inhibitor resistance mutations includeone or more RET inhibitor resistance mutations listed in Tables 3 and 4.For example, the one or more RET inhibitor resistance mutations caninclude a substitution at amino acid position 804, e.g., V804M, V804L,or V804E, or a substitution at amino acid position 810, e.g., G810S,G810R, G810C, G810A, G810V, and G810D. In some embodiments, theadditional anticancer agent is any anticancer agent known in the art.For example, the additional anticancer agent is another RET inhibitor(e.g., a second RET inhibitor). In some embodiments, the additionalanticancer agent is an immunotherapy. In some embodiments of step (c),another RET inhibitor can be the first RET inhibitor administered instep (a).

Also provided are methods of selecting a treatment for a subject havinga cancer that include (a) administering one or more doses of a first RETinhibitor to the subject for a period of time; (b) after (a),determining whether a cancer cell in a sample obtained from the subjecthas at least one RET inhibitor resistance mutation; and (c) selecting asecond RET inhibitor as a monotherapy or in conjunction with anotheranticancer agent if the subject has a cancer cell that has one or moreRET inhibitor resistance mutations; or (d) selecting additional doses ofthe first RET inhibitor of step (a) for the subject if the subject has acancer cell that does not have a RET inhibitor resistance mutation. Insome embodiments, when additional doses of the first RET inhibitor ofstep (a) are selected for the subject, the method can further includeselecting doses of another anticancer agent for the subject. In someembodiments, the one or more RET inhibitor resistance mutations conferincreased resistance to a cancer cell or tumor to treatment with thefirst RET inhibitor. In some embodiments, the one or more RET inhibitorresistance mutations include one or more RET inhibitor resistancemutations listed in Tables 3 and 4. For example, the one or more RETinhibitor resistance mutations can include a substitution at amino acidposition 804, e.g., V804M, V804L, or V804E, or a substitution at aminoacid position 810, e.g., G810S, G810R, G810C, G810A, G810V, and G810D.In some embodiments, the additional anticancer agent is any anticanceragent known in the art. For example, the additional anticancer agent isanother RET inhibitor (e.g., a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof).In some embodiments, the additional anticancer agent is animmunotherapy. In some embodiments, another RET can be the first RETinhibitor administered in step (a).

Also provided are methods of selecting a treatment for a subject havinga cancer that include (a) determining whether a cancer cell in a sampleobtained from a subject having a cancer and previously administered oneor more doses of a first RET inhibitor has one or more RET inhibitorresistance mutations; (b) selecting a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofas a monotherapy or in conjunction with another anticancer agent for thesubject if the subject has a cancer cell that has at least one RETinhibitor resistance mutation; or (c) selecting additional doses of thefirst RET inhibitor previously administered to the subject if thesubject has a cancer cell that does not have a RET inhibitor resistancemutation. In some embodiments, when additional doses of the first RETinhibitor previously administered to the subject are selected for thesubject, the method can further include selecting doses of anotheranticancer agent (e.g., a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofor immunotherapy) for the subject. In some embodiments, the one or moreRET inhibitor resistance mutations confer increased resistance to acancer cell or tumor to treatment with the first RET inhibitor. In someembodiments, the one or more RET inhibitor resistance mutations includeone or more RET inhibitor resistance mutations listed in Tables 3 and 4.For example, the one or more RET inhibitor resistance mutations caninclude a substitution at amino acid position 804, e.g., V804M, V804L,or V804E, or a substitution at amino acid position 810, e.g., G810S,G810R, G810C, G810A, G810V, and G810D. In some embodiments, theadditional anticancer agent is any anticancer agent known in the art.For example, the additional anticancer agent is another RET inhibitor(e.g., a second RET inhibitor). In some embodiments, the additionalanticancer agent is an immunotherapy. In some embodiments of step (c),another RET inhibitor can be the first RET inhibitor administered instep (a).

Also provided are methods of selecting a treatment for a subject havinga cancer that include (a) determining whether a cancer cell in a sampleobtained from a subject having a cancer and previously administered oneor more doses of a first RET inhibitor has one or more RET inhibitorresistance mutations; (b) selecting a second RET inhibitor as amonotherapy or in conjunction with another anticancer agent for thesubject if the subject has a cancer cell that has at least one RETinhibitor resistance mutation; or (c) selecting additional doses of thefirst RET inhibitor previously administered to the subject if thesubject has a cancer cell that does not have a RET inhibitor resistancemutation. In some embodiments, when additional doses of the first RETinhibitor previously administered to the subject are selected for thesubject, the method can further include selecting doses of anotheranticancer agent (e.g., a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,or an immunotherapy) for the subject. In some embodiments, the one ormore RET inhibitor resistance mutations confer increased resistance to acancer cell or tumor to treatment with the first RET inhibitor. In someembodiments, the one or more RET inhibitor resistance mutations includeone or more RET inhibitor resistance mutations listed in Tables 3 and 4.For example, the one or more RET inhibitor resistance mutations caninclude a substitution at amino acid position 804, e.g., V804M, V804L,or V804E, or a substitution at amino acid position 810, e.g., G810S,G810R, G810C, G810A, G810V, and G810D. In some embodiments, theadditional anticancer agent is any anticancer agent known in the art.For example, the additional anticancer agent is another RET inhibitor(e.g., a compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof). In some embodiments, theadditional anticancer agent is an immunotherapy. In some embodiments,another RET can be the first RET inhibitor administered in step (a).

Also provided are methods of determining a subject's risk for developinga cancer that has some resistance to a first RET inhibitor that include:determining whether a cell in a sample obtained from the subject has oneor more RET inhibitor resistance mutations; and identifying a subjecthaving a cell that has one or more RET inhibitor resistance mutations,as having an increased likelihood of developing a cancer that has someresistance to the first RET inhibitor. Also provided are methods ofdetermining a subject's risk for developing a cancer that has someresistance to a first RET inhibitor that include: identifying a subjecthaving a cell that has one or more RET inhibitor resistance mutations,as having an increased likelihood of developing a cancer that has someresistance to the first RET inhibitor. Also provided are methods ofdetermining the presence of a cancer that has some resistance to a firstRET inhibitor that include: determining whether a cancer cell in asample obtained from the subject has one or more RET inhibitorresistance mutations; and determining that the subject having a cancercell that has one or more RET inhibitor resistance mutations has acancer that has some resistance to the first RET inhibitor. Alsoprovided are methods of determining the presence of a cancer that hassome resistance to a first RET inhibitor in a subject that include:determining that a subject having a cancer cell that has one or more RETinhibitor resistance mutations, has a cancer that has some resistance tothe first RET inhibitor. In some embodiments, the one or more RETinhibitor resistance mutations confer increased resistance to a cancercell or tumor to treatment with the first RET inhibitor. In someembodiments, the one or more RET inhibitor resistance mutations includeone or more RET inhibitor resistance mutations listed in Tables 3 and 4.For example, the one or more RET inhibitor resistance mutations caninclude a substitution at amino acid position 804, e.g., V804M, V804L,or V804E, or a substitution at amino acid position 810, e.g., G810S,G810R, G810C, G810A, G810V, and G810D.

In some embodiments of any of the methods described herein, a RETinhibitor resistance mutation that confers increased resistance to acancer cell or tumor to treatment with a first RET inhibitor can be anyof the RET inhibitor resistance mutations listed in Table 3 or 4 (e.g.,a substitution at amino acid position 804, e.g., V804M, V804L, or V804E,or a substitution at amino acid position 810, e.g., G810S, G810R, G810C,G810A, G810V, and G810D).

In some embodiments, the presence of one or more RET inhibitorresistance mutations in a tumor causes the tumor to be more resistant totreatment with a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof. Methods usefulwhen a RET inhibitor resistance mutation causes the tumor to be moreresistant to treatment with a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofare described below. For example, provided herein are methods oftreating a subject having a cancer that include: identifying a subjecthaving a cancer cell that has one or more RET inhibitor resistancemutations; and administering to the identified subject a treatment thatdoes not include a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof as a monotherapy(e.g., a second RET kinase inhibitor). Also provided are methods oftreating a subject identified as having a cancer cell that has one ormore RET inhibitor resistance mutations that include administering tothe subject a treatment that does not include a compound of FormulaI-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphform thereof as a monotherapy (e.g., a second RET kinase inhibitor). Insome embodiments, the one or more RET inhibitor resistance mutationsconfer increased resistance to a cancer cell or tumor to treatment witha compound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof.

Also provided are methods of selecting a treatment for a subject havinga cancer that include: identifying a subject having a cancer cell thathas one or more RET inhibitor resistance mutations; and selecting atreatment that does not include a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofas a monotherapy for the identified subject (e.g., a second RET kinaseinhibitor). Also provided are methods of selecting a treatment for asubject having a cancer that include: selecting a treatment that doesnot include a compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof as a monotherapy (e.g., asecond RET kinase inhibitor) for a subject identified as having a cancercell that has one or more RET inhibitor resistance mutations. Alsoprovided are methods of selecting a subject having a cancer for atreatment that does not include a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofas a monotherapy (e.g., a second RET kinase inhibitor) that include:identifying a subject having a cancer cell that has one or more RETinhibitor resistance mutations; and selecting the identified subject fora treatment that does not include a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofas a monotherapy (e.g., a second RET kinase inhibitor). Also providedare methods of selecting a subject having a cancer for a treatment thatdoes not include a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof as a monotherapy(e.g., a second RET kinase inhibitor) that include: selecting a subjectidentified as having a cancer cell that has one or more RET inhibitorresistance mutations for a treatment that does not include a compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof as a monotherapy. In some embodiments, the one ormore RET inhibitor resistance mutations confer increased resistance to acancer cell or tumor to treatment with a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof.

Also provided are methods of determining the likelihood that a subjecthaving a cancer will have a positive response to treatment with acompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof as a monotherapy that include:determining whether a cancer cell in a sample obtained from the subjecthas one or more RET inhibitor resistance mutations; and determining thatthe subject having the cancer cell that has one or more RET inhibitorresistance mutations has a decreased likelihood of having a positiveresponse to treatment with a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofas a monotherapy. Also provided are methods of determining thelikelihood that a subject having cancer will have a positive response totreatment with a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof as a monotherapythat include: determining that a subject having a cancer cell that hasone or more RET inhibitor resistance mutations has a decreasedlikelihood of having a positive response to treatment with a compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof as a monotherapy. Also provided are methods ofpredicting the efficacy of treatment with a compound of Formula I-IV, ora pharmaceutically acceptable salt, amorphous, or polymorph form thereofas a monotherapy in a subject having cancer that include: determiningwhether a cancer cell in a sample obtained from the subject has one ormore RET inhibitor resistance mutations; and determining that treatmentwith a compound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof as a monotherapy is less likely tobe effective in a subject having a cancer cell in a sample obtained fromthe subject that has one or more RET inhibitor resistance mutations.Also provided are methods of predicting the efficacy of treatment with acompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof as a monotherapy in a subjecthaving cancer that include: determining that treatment with a compoundof Formula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof as a monotherapy is less likely to be effectivein a subject having a cancer cell in a sample obtained from the subjectthat has one or more RET inhibitor resistance mutations. In someembodiments, the one or more RET inhibitor resistance mutations conferincreased resistance to a cancer cell or tumor to treatment with acompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof.

Also provided are methods of treating a subject having a cancer thatinclude: (a) administering one or more doses of a compound of FormulaI-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphform thereof for a period of time; (b) after (a), determining whether acancer cell in a sample obtained from the subject has one or more RETinhibitor resistance mutations; and (c) administering a second RETinhibitor or a second compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof as a monotherapyor in conjunction with another anticancer agent to a subject having acancer cell that has one or more RET inhibitor resistance mutations; or(d) administering additional doses of the compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofof step (a) to a subject having a cancer cell that does not have a RETinhibitor resistance mutation. In some embodiments, where the subject isadministered additional doses of the compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofof step (a), the subject can also be administered another anticanceragent or a second compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof. In someembodiments, the one or more RET inhibitor resistance mutations conferincreased resistance to a cancer cell or tumor to treatment with acompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof. In some embodiments, theadditional anticancer agent is any anticancer agent known in the art.For example, the additional anticancer agent is another RET inhibitor(e.g., a second RET inhibitor). In some embodiments, the additionalanticancer agent is an immunotherapy. In some embodiments, another RETcan be the compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof administered in step (a).

Also provided are methods of treating a subject having a cancer thatinclude: (a) determining whether a cancer cell in a sample obtained froma subject having a cancer and previously administered one or more dosesof a compound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, has one or more RET inhibitorresistance mutations; (b) administering a second RET inhibitor or asecond compound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof as a monotherapy or in conjunctionwith another anticancer agent to a subject having a cancer cell that hasone or more RET inhibitor resistance mutations; or (c) administeringadditional doses of the compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof previouslyadministered to a subject having a cancer cell that does not have a RETinhibitor resistance mutation. In some embodiments, where the subject isadministered additional doses of the compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofof step (a), the subject can also be administered another anticanceragent. In some embodiments, the one or more RET inhibitor resistancemutations confer increased resistance to a cancer cell or tumor totreatment with a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof. In someembodiments, the additional anticancer agent is any anticancer agentknown in the art. For example, the additional anticancer agent isanother RET inhibitor (e.g., a second RET inhibitor). In someembodiments, the additional anticancer agent is an immunotherapy. Insome embodiments, another RET can be the compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofadministered in step (a).

Also provided are methods of selecting a treatment for a subject havinga cancer that include: (a) administering one or more doses of a compoundof Formula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof to the subject for a period of time; (b) after(a), determining whether a cancer cell in a sample obtained from thesubject has one or more RET inhibitor resistance mutations; and (c)selecting a second RET inhibitor or a second compound of Formula I-IV,or a pharmaceutically acceptable salt, amorphous, or polymorph formthereof as a monotherapy or in conjunction with another anticancer agentfor the subject if the subject has a cancer cell that has a RETinhibitor resistance mutation; or (d) selecting additional doses of thecompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof of step (a) for the subject if thesubject has a cancer cell that does not have a RET inhibitor resistancemutation. In some embodiments, where additional doses of a compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof of step (a) are selected for the subject, themethod can also include further selecting another anticancer agent. Insome embodiments, the one or more RET inhibitor resistance mutationsconfer increased resistance to a cancer cell or tumor to treatment witha compound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof. In some embodiments, theadditional anticancer agent is any anticancer agent known in the art.For example, the additional anticancer agent is another RET inhibitor(e.g., a second RET inhibitor). In some embodiments, the additionalanticancer agent is an immunotherapy. In some embodiments, another RETcan be the compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof administered in step (a).

Also provided are methods of selecting a treatment for a subject havinga cancer that include: (a) determining whether a cancer cell in a sampleobtained from a subject having a cancer and previously administered oneor more doses of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof, has one or moreRET inhibitor resistance mutations; (b) selecting a second RET inhibitoror a second compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof as a monotherapy or inconjunction with another anticancer agent for the subject if the subjecthas a cancer cell that has a RET inhibitor resistance mutation; or (c)selecting additional doses of the compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofpreviously administered to the subject if the subject has a cancer cellthat does not have a RET inhibitor resistance mutation. In someembodiments, where additional doses of the compound of Formula I-IV, ora pharmaceutically acceptable salt, amorphous, or polymorph form thereofof step (a) are selected for the subject, the method can also includefurther selecting another anticancer agent. In some embodiments, the oneor more RET inhibitor resistance mutations confer increased resistanceto a cancer cell or tumor to treatment with a compound of Formula I-IV,or a pharmaceutically acceptable salt, amorphous, or polymorph formthereof. In some embodiments, the additional anticancer agent is anyanticancer agent known in the art. For example, the additionalanticancer agent is another RET inhibitor (e.g., a second RETinhibitor). In some embodiments, the additional anticancer agent is animmunotherapy. In some embodiments, another RET can be the compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof administered in step (a).

Also provided are methods of determining a subject's risk for developinga cancer that has some resistance to a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofthat include: determining whether a cell in a sample obtained from thesubject has one or more RET inhibitor resistance mutations; andidentifying the subject if the subject has a cell that has one or moreRET inhibitor resistance mutations as having an increased likelihood ofdeveloping a cancer that has some resistance to a compound of FormulaI-IV, or a pharmaceutically acceptable salt, amorphous, or polymorphform thereof. Also provided are methods of determining a subject's riskfor developing a cancer that has some resistance to a compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof that include: identifying a subject having a cellthat has one or more RET inhibitor resistance mutations as having anincreased likelihood of developing a cancer that has some resistance toa compound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof. Also provided are methods ofdetermining the presence of a cancer that has some resistance to acompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof that includes: determining whethera cancer cell in a sample obtained from the subject has one or more RETinhibitor resistance mutations; and determining that the subject havingthe cancer cell that has one or more RET inhibitor resistance mutationshas a cancer that has some resistance to a compound of Formula I-IV, ora pharmaceutically acceptable salt, amorphous, or polymorph formthereof. Also provided are methods of determining the presence of acancer that has some resistance to a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofin a subject that include: determining that a subject having a cancercell that has one or more RET inhibitor resistance mutations has acancer that has some resistance to a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof.In some embodiments, the one or more RET inhibitor resistance mutationsconfer increased resistance to a cancer cell or tumor to treatment witha compound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof.

In some embodiments of any of the methods described herein, a RETinhibitor resistance mutation that confers increased resistance to acancer cell or tumor to treatment with a compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,can be any of the RET inhibitor resistance mutations listed in Table 3or 4.

Methods of determining the level of resistance of a cancer cell or atumor to a RET inhibitor (e.g., any of the RET inhibitors describedherein or known in the art) can be determined using methods known in theart. For example, the level of resistance of a cancer cell to a RETinhibitor can be assessed by determining the IC₅₀ of a RET inhibitor(e.g., any of the RET inhibitors described herein or known in the art)on the viability of a cancer cell. In other examples, the level ofresistance of a cancer cell to a RET inhibitor can be assessed bydetermining the growth rate of the cancer cell in the presence of a RETinhibitor (e.g., any of the RET inhibitors described herein). In otherexamples, the level of resistance of a tumor to a RET inhibitor can beassessed by determining the mass or size of one or more tumors in asubject over time during treatment with a RET inhibitor (e.g., any ofthe RET inhibitors described herein). In other examples, the level ofresistance of a cancer cell or a tumor to a RET inhibitor can beindirectly assessed by determining the activity of a RET kinaseincluding one or more of the RET inhibitor resistance mutations (i.e.,the same RET kinase expressed in a cancer cell or a tumor in a subject).The level of resistance of a cancer cell or tumor having one or more RETinhibitor resistance mutations to a RET inhibitor is relative to thelevel of resistance in a cancer cell or tumor that does not have a RETinhibitor resistance mutation (e.g., a cancer cell or tumor that doesnot have the same RET inhibitor resistance mutations, a cancer cell or atumor that does not have any RET inhibitor resistance mutations, or acancer cell or a tumor that expresses a wildtype RET protein). Forexample, the determined level of resistance of a cancer cell or a tumorhaving one or more RET inhibitor resistance mutations can be greaterthan about 1%, greater than about 2%, greater than about 3%, greaterthan about 4%, greater than about 5%, greater than about 6%, greaterthan about 7%, greater than about 8%, greater than about 9%, greaterthan about 10%, greater than about 11%, greater than about 12%, greaterthan about 13%, greater than about 14%, greater than about 15%, greaterthan about 20%, greater than about 25%, greater than about 30%, greaterthan about 35%, greater than about 40%, greater than about 45%, greaterthan about 50%, greater than about 60%, greater than about 70%, greaterthan about 80%, greater than about 90%, greater than about 100%, greaterthan about 110%, greater than about 120%, greater than about 130%,greater than about 140%, greater than about 150%, greater than about160%, greater than about 170%, greater than about 180%, greater thanabout 190%, greater than about 200%, greater than about 210%, greaterthan about 220%, greater than about 230%, greater than about 240%,greater than about 250%, greater than about 260%, greater than about270%, greater than about 280%, greater than about 290%, or greater thanabout 300% of the level of resistance in a cancer cell or tumor thatdoes not have a RET inhibitor resistance mutation (e.g., a cancer cellor tumor that does not have the same RET inhibitor resistance mutations,a cancer cell or a tumor that does not have any RET inhibitor resistancemutations, or a cancer cell or a tumor that expresses a wildtype RETprotein).

RET is thought to play an important role in the development and survivalof afferent nociceptors in the skin and gut. RET kinase knock-out micelack enteric neurons and have other nervous system anomalies suggestingthat a functional RET kinase protein product is necessary duringdevelopment (Taraviras, S. et al., Development, 1999, 126:2785-2797).Moreover population studies of patients with Hirschsprung's diseasecharacterized by colonic obstruction due to lack of normal colonicenervation have a higher proportion of both familial and sporadic lossof function RET mutations (Butler Tjaden N., et al., Transl. Res., 2013,162: 1-15). Irritable bowel syndrome (IBS) is a common illness affecting10-20% of individuals in developed countries and is characterized byabnormal bowel habits, bloating and visceral hypersensitivity(Camilleri, M., N. Engl. J. Med., 2012, 367: 1626-1635). While theetiology of IBS is unknown it is thought to result from either adisorder between the brain and gastrointestinal tract, a disturbance inthe gut microbiome or increased inflammation. The resultinggastrointestinal changes affect normal bowel transit resulting in eitherdiarrhea or constipation. Furthermore in many IBS patients thesensitization of the peripheral nervous system results in visceralhypersensitivity or allodynia (Keszthelyi, D., Eur. J. Pain, 2012, 16:1444-1454). See, e.g., U.S. Publication No. 2015/0099762.

Accordingly, provided herein are methods for treating a patientdiagnosed with (or identified as having) an irritable bowel syndrome(IBS) including diarrhea-predominant, constipation-predominant oralternating stool pattern, functional bloating, functional constipation,functional diarrhea, unspecified functional bowel disorder, functionalabdominal pain syndrome, chronic idiopathic constipation, functionalesophageal disorders, functional gastroduodenal disorders, functionalanorectal pain, and inflammatory bowel disease that includeadministering to the patient a therapeutically effective amount of acompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof.

Also provided herein are methods for treating a patient identified ordiagnosed as having a RET-associated irritable bowel syndrome (IBS)(e.g., a patient that has been identified or diagnosed as having aRET-associated irritable bowel syndrome (IBS) through the use of aregulatory agency-approved, e.g., FDA-approved, kit for identifyingdysregulation of a RET gene, a RET kinase, or expression or activity orlevel of any of the same, in a patient or a biopsy sample from thepatient) that include administering to the patient a therapeuticallyeffective amount of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof.

Also provided herein are methods for treating pain associated with IBSthat include administering to the patient a therapeutically effectiveamount of a compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof. In some embodiments, acompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof is administered in combination withanother therapeutic agent useful for treating one or more symptoms ofIBS.

Also provided are methods for treating an irritable bowel syndrome (IBS)in a patient in need thereof, the method comprising: (a) determining ifthe irritable bowel syndrome (IBS) in the patient is a RET-associatedIBS (e.g., using a regulatory-agency approved, e.g., FDA-approved, kitfor identifying dysregulation of a RET gene, a RET kinase, or expressionor activity or level of any of the same, in a patient or a biopsy samplefrom the patient, or by performing any of the non-limiting examples ofassays described herein); and (b) if the IBS is determined to be aRET-associated IBS, administering to the patient a therapeuticallyeffective amount of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof.

In some embodiments, the compounds of the present invention are usefulfor treating irritable bowel syndrome (IBS) in combination with one ormore additional therapeutic agents or therapies effective in treatingthe irritable bowel syndrome that work by the same or a differentmechanism of action. The at least one additional therapeutic agent maybe administered with a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof as part of thesame or separate dosage forms, via the same or different routes ofadministration, and on the same or different administration schedulesaccording to standard pharmaceutical practice known to one skilled inthe art.

Non-limiting examples of additional therapeutics for the treatment ofirritable bowel syndrome (IBS) include probiotics, fiber supplements(e.g., psyllium, methylcellulose), anti-diarrheal medications (e.g.,loperamide), bile acid binders (e.g., cholestyramine, colestipol,colesevelam), anticholinergic and antispasmodic medications (e.g.,hyoscyamine, dicyclomine), antidepressant medications (e.g., tricyclicantidepressant such as imipramine or notriptyline or a selectiveserotonin reuptake inhibitor (SSRI) such as fluoxetine or paroxetine),antibiotics (e.g., rifaximin), alosetron, and lubiprostone.

Accordingly, also provided herein are methods of treating irritablebowel syndrome (IBS), comprising administering to a patient in needthereof a pharmaceutical combination for treating IBS which comprises(a) a compound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, (b) an additional therapeuticagent, and (c) optionally at least one pharmaceutically acceptablecarrier for simultaneous, separate or sequential use for the treatmentof D3 S, wherein the amounts of the compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereofand the additional therapeutic agent are together effective in treatingthe IBS. In one embodiment, the compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,and the additional therapeutic agent are administered simultaneously asseparate dosages. In one embodiment, the compound of Formula I-IV, or apharmaceutically acceptable salt, amorphous, or polymorph form thereof,and the additional therapeutic agent are administered as separatedosages sequentially in any order, in jointly therapeutically effectiveamounts, e.g. in daily or intermittently dosages. In one embodiment,compound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, and the additional therapeuticagent are administered simultaneously as a combined dosage.

Also provided herein is (i) a pharmaceutical combination for treatingirritable bowel syndrome in a patient in need thereof, which comprises(a) a compound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof, (b) at least one additionaltherapeutic agent (e.g., any of the exemplary additional therapeuticagents described herein for treating irritable bowel syndrome or knownin the art), and (c) optionally at least one pharmaceutically acceptablecarrier for simultaneous, separate or sequential use for the treatmentof irritable bowel syndrome, wherein the amounts of the compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof and of the additional therapeutic agent aretogether effective in treating the irritable bowel syndrome; (ii) apharmaceutical composition comprising such a combination; (iii) the useof such a combination for the preparation of a medicament for thetreatment of irritable bowel syndrome; and (iv) a commercial package orproduct comprising such a combination as a combined preparation forsimultaneous, separate or sequential use; and to a method of treatmentof irritable bowel syndrome in a patient in need thereof. In oneembodiment the patient is a human.

The term “pharmaceutical combination”, as used herein, refers to apharmaceutical therapy resulting from the mixing or combining of morethan one active ingredient and includes both fixed and non-fixedcombinations of the active ingredients. The term “fixed combination”means that a compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof and at least one additionaltherapeutic agent (e.g., an agent effective in treating irritable bowelsyndrome), are both administered to a patient simultaneously in the formof a single composition or dosage. The term “non-fixed combination”means that a compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof and at least one additionaltherapeutic agent (e.g., an agent effective in treating irritable bowelsyndrome) are formulated as separate compositions or dosages, such thatthey may be administered to a patient in need thereof simultaneously,concurrently or sequentially with variable intervening time limits,wherein such administration provides effective levels of the two or morecompounds in the body of the patient. In one embodiment, the compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof and the additional therapeutic agent areformulated as separate unit dosage forms, wherein the separate dosagesforms are suitable for either sequential or simultaneous administration.These also apply to cocktail therapies, e.g. the administration of threeor more active ingredients.

In some embodiments, a compound provided herein can be used as an agentfor supportive care for a patient undergoing cancer treatment. Forexample, a compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof, can be useful to reduce oneor more symptoms associated with treatment with one or more cancertherapies such as diarrheal or constipations complications and/orabdominal pain. See, for example, U.S. Publication No. 2015/0099762 andHoffman, J. M. et al. Gastroenterology (2012) 142:844-854. Accordingly,a compound, or a pharmaceutically acceptable salt thereof, orcomposition provided herein can be administered to a patient to addressone or more complications associated with cancer treatment (e.g.,gastrointestinal complications such as diarrhea, constipation, orabdominal pain).

In some embodiments, a therapeutically effective amount of a compound ofFormula I-IV, or a pharmaceutically acceptable salt, amorphous, orpolymorph form thereof, can be administered to a patient undergoingcancer treatment (e.g., a patient experiencing an adverse eventassociated with cancer treatment such as an immune-related adverse eventor a gastrointestinal complication including diarrhea, constipation, andabdominal pain). For example, a compound provided herein, or apharmaceutically acceptable salt thereof, can be used in the treatmentof colitis or IBS associated with administration of a checkpointinhibitor; see, e.g., Postow, M. A. et al. Journal of Clinical Oncology(2015) 33: 1974-1982. In some such embodiments, a compound providedherein, or a pharmaceutically acceptable salt thereof, can be formulatedto exhibit low bioavailability and/or be targeted for delivery in thegastrointestinal tract. See, for example, U.S. Pat. No. 6,531,152.

Also provided is a method for inhibiting RET kinase activity in a cell,comprising contacting the cell with a compound of Formula I. In oneembodiment, the contacting is in vitro. In one embodiment, thecontacting is in vivo. In one embodiment, the contacting is in vivo,wherein the method comprises administering an effective amount of acompound of Formula I-IV, or a pharmaceutically acceptable salt,amorphous, or polymorph form thereof to a subject having a cell havingRET kinase activity. In some embodiments, the cell is a cancer cell. Inone embodiment, the cancer cell is any cancer as described herein. Insome embodiments, the cancer cell is a RET-associated cancer cell. Insome embodiments, the cell is a gastrointestinal cell.

Also provided is a method for inhibiting RET kinase activity in amammalian cell, comprising contacting the cell with a compound ofFormula I. In one embodiment, the contacting is in vitro. In oneembodiment, the contacting is in vivo. In one embodiment, the contactingis in vivo, wherein the method comprises administering an effectiveamount of a compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof to a mammal having a cellhaving RET kinase activity. In some embodiments, the mammalian cell is amammalian cancer cell. In one embodiment, the mammalian cancer cell isany cancer as described herein. In some embodiments, the mammaliancancer cell is a RET-associated cancer cell. In some embodiments, themammalian cell is a gastrointestinal cell.

As used herein, the term “contacting” refers to the bringing together ofindicated moieties in an in vitro system or an in vivo system. Forexample, “contacting” a RET kinase with a compound provided hereinincludes the administration of a compound provided herein to anindividual or patient, such as a human, having a RET kinase, as well as,for example, introducing a compound provided herein into a samplecontaining a cellular or purified preparation containing the RET kinase.

Also provided herein is a method of inhibiting cell proliferation, invitro or in vivo, the method comprising contacting a cell with aneffective amount of a compound of Formula I-IV, or a pharmaceuticallyacceptable salt, amorphous, or polymorph form thereof, or apharmaceutical composition thereof as defined herein

The phrase “effective amount” means an amount of compound that, whenadministered to a patient in need of such treatment, is sufficient to(i) treat a RET kinase-associated disease or disorder, (ii) attenuate,ameliorate, or eliminate one or more symptoms of the particular disease,condition, or disorder, or (iii) delay the onset of one or more symptomsof the particular disease, condition, or disorder described herein. Theamount of a compound of Formula I-IV, or a pharmaceutically acceptablesalt, amorphous, or polymorph form thereof that will correspond to suchan amount will vary depending upon factors such as the particularcompound, disease condition and its severity, the identity (e.g.,weight) of the patient in need of treatment, but can nevertheless beroutinely determined by one skilled in the art.

4. Pharmaceutical Compositions and Administration

When employed as pharmaceuticals, the compound of Formula I-IV,including polymorph forms and pharmaceutically acceptable salts thereof,can be administered in the form of pharmaceutical compositions. Thesecompositions can be prepared in a manner well known in thepharmaceutical art, and can be administered by a variety of routes,depending upon whether local or systemic treatment is desired and uponthe area to be treated. Administration can be topical (includingtransdermal, epidermal, ophthalmic and to mucous membranes includingintranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalationor insufflation of powders or aerosols, including by nebulizer;intratracheal or intranasal), oral or parenteral. Oral administrationcan include a dosage form formulated for once-daily or twice-daily (BID)administration. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal intramuscular or injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Parenteral administration can be in the form of a singlebolus dose, or can be, for example, by a continuous perfusion pump.Pharmaceutical compositions and formulations for topical administrationcan include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable.

Also provided herein are pharmaceutical compositions which contain, asthe active ingredient, a compound of Formula I-IV or a polymorph form orpharmaceutically acceptable salt thereof, in combination with one ormore pharmaceutically acceptable carriers (excipients). For example, apharmaceutical composition prepared using a compound of Formula I-IV ora pharmaceutically acceptable salt, amorphous, or polymorph formthereof. In some embodiments, the composition is suitable for topicaladministration. In making the compositions provided herein, the activeingredient is typically mixed with an excipient, diluted by an excipientor enclosed within such a carrier in the form of, for example, acapsule, sachet, paper, or other container. When the excipient serves asa diluent, it can be a solid, semi-solid, or liquid material, which actsas a vehicle, carrier or medium for the active ingredient. Thus, thecompositions can be in the form of tablets, pills, powders, lozenges,sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups,aerosols (as a solid or in a liquid medium), ointments containing, forexample, up to 10% by weight of the active compound, soft and hardgelatin capsules, suppositories, sterile injectable solutions, andsterile packaged powders. In some embodiments, the composition isformulated for oral administration. In some embodiments, the compositionis a solid oral formulation. In some embodiments, the composition isformulated as a tablet or capsule.

Further provided herein are pharmaceutical compositions containing acompound of Formula I-IV or a polymorph form or pharmaceuticallyacceptable salt thereof with a pharmaceutically acceptable carrier.Pharmaceutical compositions containing a compound of Formula I-IV or apolymorph form or pharmaceutically acceptable salt thereof as the activeingredient can be prepared by intimately mixing the compound of FormulaI-IV or a polymorph form or pharmaceutically acceptable salt thereofwith a pharmaceutical carrier according to conventional pharmaceuticalcompounding techniques. The carrier can take a wide variety of formsdepending upon the desired route of administration (e.g., oral,parenteral). In some embodiments, the composition is a solid oralcomposition.

Suitable pharmaceutically acceptable carriers are well known in the art.Descriptions of some of these pharmaceutically acceptable carriers canbe found in The Handbook of Pharmaceutical Excipients, published by theAmerican Pharmaceutical Association and the Pharmaceutical Society ofGreat Britain.

Methods of formulating pharmaceutical compositions have been describedin numerous publications such as Pharmaceutical Dosage Forms: Tablets,Second Edition, Revised and Expanded, Volumes 1-3, edited by Liebermanet al; Pharmaceutical Dosage Forms: Parenteral Medications, Volumes 1-2,edited by Avis et al; and Pharmaceutical Dosage Forms: Disperse Systems,Volumes 1-2, edited by Lieberman et al; published by Marcel Dekker, Inc.

In preparing the compositions in oral dosage form, any of the usualpharmaceutical media can be employed. Thus for liquid oral preparationssuch as suspensions, elixirs and solutions, suitable carriers andadditives include water, glycols, oils, alcohols, flavoring agents,preservatives, stabilizers, coloring agents and the like; for solid oralpreparations, such as powders, capsules and tablets, suitable carriersand additives include starches, sugars, diluents, granulating agents,lubricants, binders, disintegrating agents and the like. Suitablebinders include, without limitation, starch, gelatin, natural sugarssuch as glucose or beta-lactose, corn sweeteners, natural and syntheticgums such as acacia, tragacanth or sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like. Disintegrators include, without limitation, starch, methylcellulose, agar, bentonite, xanthan gum and the like. Solid oralpreparations can also be coated with substances such as sugars or beenteric-coated so as to modulate major site of absorption. Forparenteral administration, the carrier will usually consist of sterilewater and other ingredients can be added to increase solubility orpreservation. Injectable suspensions or solutions can also be preparedutilizing aqueous carriers along with appropriate additives. Thepharmaceutical compositions herein will contain, per dosage unit, e.g.,tablet, capsule, powder, injection, teaspoonful and the like, an amountof the active ingredient necessary to deliver an effective dose asdescribed herein.

The compositions comprising a compound of Formula I-IV or a polymorphform or pharmaceutically acceptable salt thereof can be formulated in aunit dosage form, each dosage containing from about 5 to about 1,000 mg(1 g), more usually about 100 mg to about 500 mg, of the activeingredient. The term “unit dosage form” refers to physically discreteunits suitable as unitary dosages for human subjects and other patients,each unit containing a predetermined quantity of active material (i.e.,a compound of Formula I-IV or a polymorph form or pharmaceuticallyacceptable salt thereof) calculated to produce the desired therapeuticeffect, in association with a suitable pharmaceutical excipient.

In some embodiments, the compositions provided herein contain from about5 mg to about 50 mg of the active ingredient. One having ordinary skillin the art will appreciate that this embodies compounds or compositionscontaining about 5 mg to about 10 mg, about 10 mg to about 15 mg, about15 mg to about 20 mg, about 20 mg to about 25 mg, about 25 mg to about30 mg, about 30 mg to about 35 mg, about 35 mg to about 40 mg, about 40mg to about 45 mg, or about 45 mg to about 50 mg of the activeingredient.

In some embodiments, the compositions provided herein contain from about50 mg to about 500 mg of the active ingredient. One having ordinaryskill in the art will appreciate that this embodies compounds orcompositions containing about 50 mg to about 100 mg, about 100 mg toabout 150 mg, about 150 mg to about 200 mg, about 200 mg to about 250mg, about 250 mg to about 300 mg, about 350 mg to about 400 mg, or about450 mg to about 500 mg of the active ingredient. In some embodiments,the compositions provided herein contain about 10 mg, about 20 mg, about80 mg, or about 160 mg of the active ingredient.

In some embodiments, the compositions provided herein contain from about500 mg to about 1,000 mg of the active ingredient. One having ordinaryskill in the art will appreciate that this embodies compounds orcompositions containing about 500 mg to about 550 mg, about 550 mg toabout 600 mg, about 600 mg to about 650 mg, about 650 mg to about 700mg, about 700 mg to about 750 mg, about 750 mg to about 800 mg, about800 mg to about 850 mg, about 850 mg to about 900 mg, about 900 mg toabout 950 mg, or about 950 mg to about 1,000 mg of the activeingredient.

The daily dosage of the compound of Formula I-IV or a polymorph form orpharmaceutically acceptable salt thereof can be varied over a wide rangefrom 1.0 to 10,000 mg per adult human per day, or higher, or any rangetherein. For oral administration, the compositions are preferablyprovided in the form of tablets containing, 0.01, 0.05, 0,1, 0.5, 1.0,2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 150, 160, 200, 250 and 500milligrams of the active ingredient for the symptomatic adjustment ofthe dosage to the patient to be treated. An effective amount of the drugis ordinarily supplied at a dosage level of from about 0.1 mg/kg toabout 1000 mg/kg of body weight per day, or any range therein.Preferably, the range is from about 0.5 to about 500 mg/kg of bodyweight per day, or any range therein. More preferably, from about 1.0 toabout 250 mg/kg of body weight per day, or any range therein. Morepreferably, from about 0.1 to about 100 mg/kg of body weight per day, orany range therein. In an example, the range can be from about 0.1 toabout 50.0 mg/kg of body weight per day, or any amount or range therein.In another example, the range can be from about 0.1 to about 15.0 mg/kgof body weight per day, or any range therein. In yet another example,the range can be from about 0.5 to about 7.5 mg/kg of body weight perday, or any amount to range therein. Pharmaceutical compositionscontaining a compound of Formula I-IV or a polymorph form orpharmaceutically acceptable salt thereof can be administered on aregimen of 1 to 4 times per day or in a single daily dose.

The active compound may be effective over a wide dosage range and isgenerally administered in a pharmaceutically effective amount. Optimaldosages to be administered can be readily determined by those skilled inthe art. It will be understood, therefore, that the amount of thecompound actually administered will usually be determined by aphysician, and will vary according to the relevant circumstances,including the mode of administration, the actual compound administered,the strength of the preparation, the condition to be treated, and theadvancement of the disease condition. In addition, factors associatedwith the particular patient being treated, including; patient response,age, weight, diet, time of administration and severity of the patient'ssymptoms, will result in the need to adjust dosages.

In some embodiments, the compounds provided herein can be administeredin an amount ranging from about 1 mg/kg to about 100 mg/kg. In someembodiments, the compound provided herein can be administered in anamount of about 1 mg/kg to about 20 mg/kg, about 5 mg/kg to about 50mg/kg, about 10 mg/kg to about 40 mg/kg, about 15 mg/kg to about 45mg/kg, about 20 mg/kg to about 60 mg/kg, or about 40 mg/kg to about 70mg/kg. For example, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg,about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg. In someembodiments, such administration can be once-daily or twice-daily (BID)administration.

In some embodiments, the compounds provided herein can be administeredin an amount of about 10 mg twice a day (BID), 20 mg BID, about 40 mgBID, about 60 mg BID, about 80 mg BID, about 120 mg BID, about 160 mgBID, and about 240 mg BID. In some embodiments, each dose isadministered at least six hours after the previous dose. In someembodiments, each dose is administered at least twelve hours after theprevious dose.

In some embodiments, a compound of Formula I-IV, or a polymorph form orpharmaceutically acceptable salt thereof exhibits pH dependentsolubility at lower pH values. Accordingly, patients also receivingproton pump inhibitors (PPIs) and/or antacids may need to adjust thedosage of the compound of Formula I-IV, or a polymorph form orpharmaceutically acceptable salt thereof (e.g., increase the dose of thecompound of Formula I-IV, or a polymorph form or pharmaceuticallyacceptable salt thereof). In some embodiments, the isoform of cytochromeP450 (CUP) that metabolizes a compound of Formula I-IV, or a polymorphform or pharmaceutically acceptable salt thereof,ï is CYP3A4.Accordingly, patients also receiving agents that inhibit or induceCYP3A4 may need to adjust the dosage of the compound of Formula I-IV, ora polymorph form or pharmaceutically acceptable salt thereof (e.g.,increase the dose of the compound of Formula I-IV, or a polymorph formor pharmaceutically acceptable salt thereof, in the case of a CYP3A4inducer or decrease the dose of the compound of Formula I-IV, or apolymorph form or pharmaceutically acceptable salt thereof, in the caseof a CYP3A4 inhibitor).

One skilled in the art will recognize that both in vivo and in vitrotrials using suitable, known and generally accepted cell and/or animalmodels are predictive of the ability of a test compound to treat orprevent a given disorder.

One skilled in the art will further recognize that human clinical trialsincluding first-in-human, dose ranging and efficacy trials, in healthypatients and/or those suffering from a given disorder, can be completedaccording to methods well known in the clinical and medical arts.

5. Kits

Provided herein are pharmaceutical kits useful, for example, in thetreatment of RET-associated diseases or disorders, such as cancer orirritable bowel syndrome (IBS), which include one or more containerscontaining a pharmaceutical composition comprising a therapeuticallyeffective amount of a compound provided herein. Such kits can furtherinclude, if desired, one or more of various conventional pharmaceuticalkit components, such as, for example, containers with one or morepharmaceutically acceptable carriers, additional containers, etc., aswill be readily apparent to those skilled in the art. Instructions,either as inserts or as labels, indicating quantities of the componentsto be administered, guidelines for administration, and/or guidelines formixing the components, can also be included in the kit.

EXAMPLES

The following examples illustrate the invention.

Example 1: Synthesis of the Compound of Formula I Intermediate A1 and A26-bromo-4-methoxypyrazolo[1,5-a]pyridine (A1) and4-bromo-6-methoxypyrazolo[1,5-a]pyridine (A2)

Part A: Preparation of O-(mesitylsulfonyl)hydroxylamine (IntermediateR1) Step 1: Preparation of tert-butyl (mesitylsulfonyl)oxycarbamate

To a 0° C. solution of 2,4,6-trimethylbenzene-1-sulfonyl chloride (10.0g, 45.72 mmol) and tert-butyl hydroxycarbamate (6.088 g, 45.72 mmol) inMTBE (100 mL) was added TEA (14.46 mL, 48.01 mmol) dropwise whilestirring. The resulting suspension was stirred at 0° C. for anadditional 30 min and then warmed to ambient temperature. The reactionwas then diluted with water (100 mL) and adjusted to pH 4 with 1 NHCl_((aq)). The organic layer was dried (Na₂SO₄), filtered, andconcentrated to yield the title compound initially as a yellowish oil,which upon drying overnight under high vacuum became a white solid(12.89 g, 89% yield). 41 NMR (CDCl₃): δ 7.66 (br s, 1H), 6.98 (s, 2H),2.67 (s, 6H), 2.32 (s, 3H), 1.31 (s, 9H).

Step 2: Preparation of O-(mesitylsulfonyl)hydroxylamine (IntermediateR1)

To TFA (117 mL, 1521 mmol) at 0° C. was slowly added tert-butyl(mesitylsulfonyl)oxycarbamate (39.0 g, 124 mmol) over 25 min. Thereaction mixture was stirred at 0° C. for 1.5 h and then quenched withthe sequential addition of crushed ice and water. The resulting thicksuspension was vigorously stirred at ambient temperature for 5 min.Without allowing the filter cake to run dry, the solids were collectedby careful vacuum filtration, followed by subsequent rinsing with water(4 L) until the filtrate reached pH 6 (Caution: explosion risk existswith dry compound at ambient temperature). The wet filter cake was takenup in dichloromethane (150 mL) and the resulting biphasic solution wasseparated. The dichloromethane layer was dried over MgSO₄ for 30 min andthen filtered and rinsed with dichloromethane (420 mL) to provide thetitle compound as a 0.22 M solution in dichloromethane.

Part B: Preparation of 6-bromo-4-methoxypyrazolo[1,5-a]pyridine (A1) and4-bromo-6-methoxypyrazolo[1,5-a]pyridine (A2) Step 1: Preparation of1-amino-3-bromo-5-methoxypyridin-1-ium 2,4,6-trimethylbenzenesulfonate

To a solution of O-(mesitylsulfonyl)hydroxylamine (Intermediate R1)(26.6 g, 117 mmol) in DCM (570 mL) cooled to 0° C. was added3-bromo-5-methoxypyridine (22.1 g, 117 mmol) in portions. The reactionmixture was stirred for 1 h at 0° C. then treated with additional3-bromo-5-methoxypyridine (250 mg, 1.39 mmol) and stirred for anadditional 2 h at 0° C. The reaction mixture was diluted with Et₂O (600mL), stirred at 0° C. for 10 min and then vacuum filtered, rinsed withEt₂O (3×250 mL). Upon reduction in volume by about ⅓, the filtrateyielded additional precipitate which was collected by filtration. Bothfilter cakes were dried in vacuo to provide the title compound (39.3 g,83% yield). ¹H NMR (CDCl₃) δ 9.25 (br s, 1H), 8.99 (m, 1H), 8.74 (m,1H), 7.46 (m, 1H), 6.83 (s, 2H), 3.92 (s, 3H), 2.65 (s, 6H), 2.22 (s,3H).

Step 2: Preparation of Ethyl6-bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carboxylate and Ethyl4-bromo-6-methoxypyrazolo[1,5-a]pyridine-3-carboxylate

To a magnetically stirred white suspension of1-amino-3-bromo-5-methoxypyridin-1-ium 2,4,6-trimethylbenzenesulfonate(33.24 g, 82.42 mmol) in DMF (82 mL) at ambient temperature was addedTEA (22.98 mL, 164.8 mmol), followed by drop-wise addition of ethylpropiolate (16.71 mL, 164.8 mmol). After vigorous stirring for 2 d, thereaction was slowly quenched via portion-wise addition to rapidlystirring ice water (820 mL). The mixture was stirred at ambienttemperature for 10 min and then vacuum filtered. Solids collected wererinsed with water and air-dried, yielding the title compounds as anorange solid in an isomeric ratio of about 4:1 (by ¹H NMR) with the 6-Brisomer as the major isomer (21 g). The wet solid isomeric mixture (about75% w/w) was directly used in Step 3 without further purification. MS(apci) m/z=298.9, 300.9 (M+H). Regioisomeric ratio was determined by MeOchemical shift in ¹H NMR (CDCl₃) δ 3.98 (6-Br isomer) vs. 3.83 (4-Brisomer).

Step 3: Preparation of 6-bromo-4-methoxypyrazolo[1,5-a]pyridine (A1) and4-bromo-6-methoxypyrazolo[1,5-a]pyridine (A2)

The isomeric mixture of ethyl6-bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carboxylate and ethyl4-bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carboxylate from Step 2 (15g, 50.1 mmol) was added to 48% HBr (114 mL) while stirring, then heatedat 80° C. for 90 min followed by stirring at ambient temperatureovernight. The resulting suspension was vacuum filtered and rinsed withwater. The aqueous filtrate and the filter cake were treatedindependently. The filter cake was taken up in MTBE and vacuum filteredto remove insoluble impurities. The MTBE filtrate was dried overanhydrous Na₂SO₄, filtered and concentrated in vacuo to yield6-bromo-4-methoxypyrazolo[1,5-a]pyridine (Intermediate A1) as a beigesolid (about 98:2 6-/4-Br; 5.08 g). MS (apci) m/z=226.9, 228.9 (M+H). ¹HNMR (CDCl₃) δ 8.26 (m, 1H), 7.82 (d, 1H), 6.61 (m, 1H), 6.43 (m, 1H),3.94 (s, 3H).

Independently the original aqueous reaction mixture filtrate wasextracted with EtOAc (2×500 mL). The combined organic extracts weredried (Na₂SO₄), filtered and concentrated in vacuo. The crude residuewas taken up in DCM (50 mL) and then filtered to remove insolublesolids. Concentration of the DCM filtrate under vacuum followed bysilica chromatography (0 to 50% EtOAc/hexanes) yielded a second batch of6-bromo-4-methoxypyrazolo[1,5-a]pyridine (Intermediate A1) as whitesolid (upper R_(f) spot, 2.06 g), as well as the minor isomer titlecompound 4-bromo-6-methoxypyrazolo[1,5-a]pyridine (Intermediate A2) alsoas white solid (lower R_(f) spot, 1.32 g). MS (apci) m/z=226.9, 228.9(M+H). ¹H NMR (CDCl₃) δ 8.02 (m, 1H), 7.85 (d, 1H), 7.17 (d, 1H), 6.55(m, 1H), 3.80 (s, 3H).

Intermediate A3

6-bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carbaldehyde

To a 0° C. solution of 6-bromo-4-methoxypyrazolo[1,5-a]pyridine(Intermediate A1, 0.75 g, 3.303 mmol) in DMF (33 mL) was slowly addedPOCl₃ (0.92 mL, 9.909 mmol). The reaction was warmed to ambienttemperature and stirred for 4 h and then diluted with H₂O (30 mL). Theresulting suspension was basified to pH 9-10 with 1 M NaOH_((aq)), thenstirred for 1 h and vacuum filtered, then rinsed sequentially with H₂O(25 mL) and MTBE (50 mL) to yield the title compound (0.76 g, 90%yield). MS (apci) m/z=256.9 (M+H).

Intermediate A4

Step 1: Preparation of(E)-6-bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carbaldehyde oxime

To a suspension of6-Bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carbaldehyde (IntermediateA3, 0.76 g, 3.0 mmol) and hydroxylamine hydrochloride (0.31 g, 4.5 mmol)in EtOH (40 mL) was added water (20 mL), and the reaction was stirred at50° C. for 4 h. After cooling to ambient temperature the reactionmixture was concentrated in vacuo. The residue was suspended in water,then treated with saturated NaHCO_(3(aq)) and vacuum filtered. Thesolids were rinsed sequentially with H₂O (25 mL) and MTBE (50 mL) toyield the title compound (0.68 g, 84% yield). MS (apci) m/z=271.9 (M+H).

Step 2: Preparation of6-bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carbonitrile

A solution of(E)-6-bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carbaldehyde oxime (17.15g, 63.50 mmol) in acetic anhydride (707 mL, 7.49 mol) was heated at 120°C. overnight. Following subsequent distillation to remove the aceticanhydride, the remaining residue was dried in vacuo to yield the titlecompound (15.92 g, 99.4% yield). ¹H NMR (CDCl₃) δ 8.32 (m, 1H), 8.12 (s,1H), 6.74 (m, 1H), 4.03 (s, 3H).

Intermediate A5

3-cyano-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-4-yltrifluoromethanesulfonate Step 1: Preparation of4-methoxy-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile

To a solution of 6-bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carbonitrile(Intermediate A4, 50 g, 198.4 mmol) and1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole(49.53 g, 238.0 mmol) in dioxane (660 mL) was added 2 M Na₂CO_(3(aq))(297.5 mL, 595.1 mmol). The reaction mixture was sparged with nitrogenfor 20 min before Pd(PPh₃)₄ (4.584 g, 3.967 mmol) was introduced,followed by additional 5 min of sparging with nitrogen. The reaction washeated at 80° C. for 18 h, then cooled to ambient temperature andvigorously stirred for 2 h. The suspension was vacuum filtered, rinsedsequentially with H₂O (2×300 mL) and MTBE (3×300 mL), then dried invacuo overnight to yield the title compound, which was used in the nextstep without further purification (52.62 g). MS (apci), m/z=254.1 (M+H).

Step 2: Preparation of4-hydroxy-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile

To a suspension of4-methoxy-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile(52.62 g, 207.8 mmol) in DCE (2 L) was added AlCl₃ (92.86 g, 696.42mmol), and the reaction mixture was stirred at 80° C. for 3 h.Additional AlCl₃ (2.5 g, 18.75 mmol) was introduced and the reaction wasrefluxed overnight. After cooling to ambient temperature the reactionmixture was diluted with DCE (1 L) and then quenched with portions ofH₂O (5×500 mL). The mixture was stirred at ambient temperature for 3 hbefore the resulting suspension was vacuum filtered and the filter cakedried in a vacuum oven (40° C.) to afford the title compound, which wasused in the next step without further purification (43.69 g). MS (apci)m/z=239.9 (M+H). ¹H NMR (d⁶-DMSO) δ 11.38 (s, 1H), 8.74 (d, 1H), 8.50(s, 1H), 8.21 (s, 1H), 7.94 (s, 1H), 6.96 (d, 1H), 3.88 (s, 3H).

Step 3: Preparation of3-cyano-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-4-yltrifluoromethanesulfonate

To a suspension of4-hydroxy-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile(43.69 g, 182.6 mmol) in DMA (365 mL) was added DIEA (63.6 mL, 365.3mmol) followed by1,1,1-trifluoro-N-phenyl-N-((trifluoromethyl)sulfonyl)methanesulfonamide(71.77 g, 200.9 mmol). The resulting solution was stirred at ambienttemperature for 2 h and then slowly poured into H₂O (4 L). The resultingsuspension was stirred for 2 h then vacuum filtered. The filter cake wasrinsed with H₂O (3×500 mL) and air dried overnight. The filter cake wasthen dissolved in DCM (1.6 L) and the resulting biphasic mixture wasphase-separated. The organic layer was dried over anhydrous MgSO₄,filtered through Celite® and rinsed with DCM. The combined organiclayers were concentrated to yield the title compound as a 90% pure tansolid (64.3 g, 95% yield). The purity of the title compound can befurther improved to >95% via silica chromatography (0-90%acetone/hexanes). ¹⁹F NMR (CDCl₃) δ −72.0. ¹H NMR (CDCl₃) δ 8.66 (d,1H), 8.29 (s, 1H), 7.77 (d, 1H), 7.70 (s, 1H), 7.55 (d, 1H), 4.01 (s,3H).

Intermediate A6

tert-butyl4-(5-(3-cyano-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-4-yl)pyridin-2-yl)piperazine-1-carboxylate

To a mixture of3-cyano-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-4-yltrifluoromethanesulfonate (Intermediate A5; 10.0 g, 26.9 mmol) andtert-butyl4-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)piperazine-1-carboxylate(12.6 g, 32.3 mmol) in dioxane (250 mL) was added 2 M Na₂CO_(3(aq))(14.3 g, 135 mmol), and the reaction mixture was sparged with nitrogenfor 15 min before introducing Pd₂(dba)₃ (1.23 g, 1.35 mmol) and X-Phos(2.57 g, 5.39 mmol). The mixture was sparged with nitrogen for anadditional 5 min and then heated at 80° C. overnight. After cooling toambient temperature, the reaction mixture was poured into H₂O (1.5 L)and stirred for 2 h. The resulting suspension was filtered and rinsedsequentially with H₂O (3×200 mL), MTBE (4×100 mL) and hexanes (4×100mL), yielding the title compound as a solid after drying in vacuoovernight (12 g, 92% yield). MS (apci) m/z=485.2 (M+H).

Intermediate A7

6-(1-methyl-1H-pyrazol-4-yl)-4-(6-(piperazin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitriledihydrochloride

To a solution of tert-butyl4-(5-(3-cyano-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-4-yl)pyridin-2-yl)piperazine-1-carboxylate(A6, 12.0 g, 24.77 mmol) in MeOH (12 mL) and DCM (50 mL) was added HCl(5-6M in iPrOH, 49.53 mL, 247.7 mmol). After stirring at ambienttemperature for 21 h the reaction was diluted with MeOH (50 mL) and DCM(50 mL). The suspension was stirred at ambient temperature until LCMSindicated the reaction was complete. The reaction mixture was filtered,rinsed with Et₂O (5×50 mL) and then dried for 19 h in a 45° C. vacuumoven to yield the title compound (9.97 g, 88% yield). MS (apci)m/z=385.1 (M+H).

Compound of Formula I

4-(6-(4-(6-methoxypyridin-3-yl)methyl)piperazin-1-yl)pyridin-3-yl)-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile

Procedure 1:

A room temperature solution of6-(1-methyl-1H-pyrazol-4-yl)-4-(6-(piperazin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitriledihydrochloride (A7; 0.125 g, 0.273 mmol) in dry DMA (2.5 mL) wastreated with TEA (114 μL, 0.820 mmol), Me₄N(AcO)₃BH (144 mg, 0.547 mmol)and 6-methoxynicotinaldehyde (0.0750 g, 0.547 mmol). The resultingmixture was stirred overnight at room temperature, then quenched withwater and CHCl₃ and allowed to stir for 30 min. The resulting biphasicmixture was filtered through a PS frit, and the aqueous layer was washedwith CHCl₃. The organic filtrate was concentrated in vacuo, and theresidue was purified by C18 reverse-phase chromatography (5-90%ACN/water as the gradient eluent) to afford the title compound (56 mg,41% yield). MS (apci) m/z=506.0 (M+H).

Procedure 2:

To a reaction vessel was charged6-(-1-methyl-1H-pyrazol-4-yl)-4-(6-(piperazin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitriletetrahydrochloride (A7; 19.0 g), and 6-methoxynicotinaldehyde (7.37 g).DMSO (247 mL) was added followed by triethylamine (15 mL). The yellowslurry stirred at room temperature for ˜2.5 hours and then sodiumtriacetoxyborohydride (15.2 g) was added in one portion and the reactionwas heated to 30° C. and stirred overnight. The reaction was judgedcomplete by HPLC and the mixture was cooled in an ice/water bath to 19°C. Water (550 mL) was added slowly, keeping the temperature below 30° C.The suspension stirred for 3.5 hours and was then filtered and the cakewas washed with water (2×285 mL). The solid was dried in a vacuum ovenat 45° C. to produce the title compound (18.0 g, 99.4%). The crystallinesolid was analyzed by XRPD producing a diffraction pattern consistentwith Formula I form A.

Example 2: Synthesis of Compounds of Formula II-IV Intermediate B1

4-Bromo-6-hydroxypyrazolo[1,5-a]pyridine-3-carbonitrile Step 1:Preparation of1-amino-3-bromo-5-methoxypyridin-1-ium-2,4,6-trimethylbenzenesulfonate

To a solution of O-(mesitylsulfonyl)hydroxylamine (Intermediate R1, 26.6g, 117 mmol) in dichloromethane (570 mL) cooled to 0° C. was added3-bromo-5-methoxypyridine (22.1 g, 117 mmol) in portions. The reactionmixture was stirred for 1 h at 0° C. then treated with additional3-bromo-5-methoxypyridine (250 mg, 1.39 mmol) and stirred for anadditional 2 h at 0° C. The reaction mixture was diluted with Et₂O (600mL), stirred at 0° C. for 10 min and then vacuum filtered, and rinsedwith Et₂O (3×250 mL). Upon reduction in volume by about ⅓, the filtrateyielded additional precipitate which was collected by filtration. Bothfilter cakes were dried in vacuo to provide the title compound (39.3 g,83% yield). ¹H NMR (CDCl₃): δ 9.25 (br s, 1H), 8.99 (m, 1H), 8.74 (m,1H), 7.46 (m, 1H), 6.83 (s, 2H), 3.92 (s, 3H), 2.65 (s, 6H), 2.22 (s,3H).

Step 2: Preparation ofethyl-6-bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carboxylate andethyl-4-bromo-6-methoxypyrazolo[1,5-a]pyridine-3-carboxylate

To a magnetically stirred white suspension of1-amino-3-bromo-5-methoxypyridin-1-ium-2,4,6-trimethylbenzenesulfonate(33.24 g, 82.42 mmol) in DMF (82 mL) at ambient temperature was addedTEA (22.98 mL, 164.8 mmol), followed by dropwise addition of ethylpropiolate (16.71 mL, 164.8 mmol). After vigorous stirring for 2 d, thereaction was slowly quenched via portion-wise addition to rapidlystirring ice water (820 mL). The mixture was stirred at ambienttemperature for 10 min and then vacuum filtered. Solids collected wererinsed with water and air-dried, yielding the title compounds as anorange solid in an isomeric ratio of about 4:1 (by ¹H NMR) with the 6-Brisomer as the major isomer (21 g). The wet solid isomeric mixture (about75% w/w) was directly used in Step 3 without further purification. MS(apci) m/z=298.9, 300.9 (M+H). Regioisomeric ratio was determined by MeOchemical shift in ¹H NMR (CDCl₃) δ 3.98 (6-Br isomer) vs. 3.83 (4-Brisomer).

Step 3: Preparation of 6-bromo-4-methoxypyrazolo[1,5-a]pyridine(Intermediate B1) and 4-bromo-6-methoxypyrazolo[1,5-a]pyridine

The isomeric mixture ofethyl-6-bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carboxylate andethyl-4-bromo-6-methoxypyrazolo[1,5-a]pyridine-3-carboxylate from Step 2(15 g, 50.1 mmol) was added to 48% HBr (114 mL) while stirring, thenheated at 80° C. for 90 min, followed by stirring at ambient temperatureovernight. The resulting suspension was vacuum filtered and rinsed withwater. The aqueous filtrate and the filter cake were treatedindependently. The filter cake was taken up in MTBE and vacuum filteredto remove insoluble impurities. The MTBE filtrate was dried overanhydrous Na₂SO₄, filtered and concentrated in vacuo to yield6-bromo-4-methoxypyrazolo[1,5-a]pyridine as a beige solid (about 98:26-/4-Br; 5.08 g). MS (apci) m/z=226.9, 228.9 (M+H). ¹H NMR (CDCl₃): δ8.26 (m, 1H), 7.82 (d, 1H), 6.61 (m, 1H), 6.43 (m, 1H), 3.94 (s, 3H).Independently, the original aqueous reaction mixture filtrate wasextracted with EtOAc (2×500 mL). The combined organic extracts weredried (Na₂SO₄), filtered and concentrated in vacuo. The crude residuewas taken up in DCM (50 mL) and then filtered to remove insolublesolids. Concentration of the DCM filtrate under vacuum followed bysilica chromatography (0 to 50% EtOAc/hexanes) yielded a second batch of6-bromo-4-methoxypyrazolo[1,5-a]pyridine (Intermediate 1) as a whitesolid (upper R_(f) spot, 2.06 g), as well as the minor isomer titlecompound 4-bromo-6-methoxypyrazolo[1,5-a]pyridine also as a white solid(lower R_(f) spot, 1.32 g). MS (apci) m/z=226.9, 228.9 (M+H). ¹H NMR(CDCl₃): δ 8.02 (m, 1H), 7.85 (d, 1H), 7.17 (d, 1H), 6.55 (m, 1H), 3.80(s, 3H).

Step 4: Preparation of4-bromo-6-methoxypyrazolo[1,5-a]pyridine-3-carbaldehyde

A solution of 4-bromo-6-methoxypyrazolo[1,5-a]pyridine (5.0 g, 22 mmol)in DMF (220 mL) was cooled to 0° C. and then slowly treated with POCl₃(6.2 mL, 66 mmol). The reaction was warmed to ambient temperature andstirred overnight. The reaction mixture was cooled to 0° C., quenchedwith water (220 mL), and basified with 6 M NaOH_((aq)) to pH 9-10. Thereaction mixture was stirred for 1 h and then vacuum filtered. Thesolids were rinsed sequentially with water (3×50 mL) and MTBE (3×50 mL).The collected solid was suspended in DCM (500 mL) and stirred in asonicating bath for 30 min and then vacuum filtered. The filtrate wasretained, while the filter cake was taken up in water (300 mL) andextracted with DCM. The organic extracts, along with the retained DCMfiltrate, were combined and dried over anhydrous Na₂SO₄, then filteredand concentrated in vacuo to provide the title compound (4.84 g, 86%yield). MS (apci), m/z=256.9 (M+H).

Step 5: Preparation of4-bromo-6-methoxypyrazolo[1,5-a]pyridine-3-carbaldehyde oxime

To a suspension of4-bromo-6-methoxypyrazolo[1,5-a]pyridine-3-carbaldehyde (4.84 g, 19.0mmol) in EtOH (253 mL) at ambient temperature was added water (127 mL)and hydroxylamine hydrochloride (1.98 g, 28.5 mmol). After stirring at50° C. overnight, the reaction mixture was cooled to ambient temperatureand concentrated in vacuo. The residue was suspended in water (150 mL)and then quenched slowly with saturated NaHCO_(3(aq)) (30 mL). Afterstirring for 1 hour at ambient temperature, the suspension was vacuumfiltered and the filter cake rinsed sequentially with H₂O (500 mL) andMTBE (100 mL) to yield the title compound as a 2:1 E/Z mixture (5.13 g,quantitative yield), which was used in the next step without furtherpurification. MS (apci) m/z=271.9 (M+H).

Step 6: Preparation of4-bromo-6-methoxypyrazolo[1,5-a]pyridine-3-carbonitrile

The E/Z mixture of4-bromo-6-methoxypyrazolo[1,5-a]pyridine-3-carbaldehyde oxime (4.95 g,18.33 mmol) in acetic anhydride (172.9 mL, 1833 mmol) was stirred at140° C. for 25 h, and then cooled to ambient temperature. The resultingsuspension was further cooled in an ice bath for 15 min and then vacuumfiltered and rinsed sequentially with water (200 mL) and MTBE (300 mL)to provide the title compound (3.74 g, 81% yield). ¹H NMR (d⁶-DMSO): δ8.70 (s, 1H), 8.60 (s, 1H), 7.78 (s, 1H), 3.83 (s, 3H).

Step 7: Preparation of4-bromo-6-hydroxypyrazolo[1,5-a]pyridine-3-carbonitrile

A slurry of 4-bromo-6-methoxypyrazolo[1,5-a]pyridine-3-carbonitrile(50.0 g, 198.4 mmol) in DCE (500 mL) was treated with AlCl₃ (79.34 g,595.1 mmol). Under an N_(2(g)) atmosphere, the resulting mixture wasstirred 19 h at 76° C., before cooling to room temperature. Using THF(1750 mL) as a rinse solvent, the reaction mixture was poured into amechanically stirred suspension of sodium sulfate decahydrate (10 eq,639 g) in THF (1000 mL). After stirring overnight at ambienttemperature, the resulting suspension was filtered, and the solids wererinsed with additional THF (2×250 mL). The filtrate was concentrated invacuo, and the resulting solid was dried under high vacuum for 3 days toafford the title compound (46.18 g, 98% yield) in sufficient purity forsubsequent use. ¹H NMR (d⁶-DMSO): δ 10.48 (s, 1H), 8.58 (s, 1H), 8.38(d, 1H), 7.64 (3, 1H).

Intermediate B2

4-bromo-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridine-3-carbonitrile

In a pressure vessel, a mixture of4-bromo-6-hydroxypyrazolo[1,5-a]pyridine-3-carbonitrile (IntermediateB1; 10.0 g, 42.0 mmol) and K₂CO₃(s) (17.4 g, 126 mmol) in DMF (50 mL)was treated with 2,2-dimethyloxirane (36.9 mL, 420 mmol). After sealingthe vessel, the reaction mixture was stirred for 12 h at 60° C., thenfor 12 h at 85° C. The mixture was allowed to cool to ambienttemperature. The room temperature mixture was poured into water (400mL), then stirred for 1 hour at ambient temperature. The resultantsuspension was vacuum filtered and the filter cake was rinsed withwater. The solids were collected and dried in vacuo to cleanly providethe title compound (11 g, 84% yield).

Intermediate B3

4-(6-fluoropyridin-3-yl)-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridine-3-carbonitrile

A mixture of4-bromo-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridine-3-carbonitrile(Intermediate B2; 10.0 g, 32.2 mmol),2-fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (10.8g, 48.4 mmol) and Pd(PPh₃)₄ (1.12 g, 0.967 mmol) in dioxane (200 mL) wastreated with 2 M Na₂CO_(3(aq)) (64.5 mL, 129 mmol). The resultingmixture was sparged with Ar_((g)), then stirred for 12 h at 85° C. underan atmosphere of N_(2(g)). After cooling to ambient temperature, theresultant mixture was poured into cold water (1.5 L). The pH of themixture was adjusted to about pH 6 with the addition of 10% citric acid.After stirring for 1 hour at ambient temperature, the resultantsuspension was vacuum filtered. The solids were collected and dried invacuo to cleanly provide the title compound (10 g, 95% yield).

Intermediate B4

4-(6-(3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridine-3-carbonitriledihydrochloride Step 1: Preparation oftert-butyl-3-(5-(3-cyano-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridin-4-yl)pyridin-2-yl)-3,6-diazabicyclo[3.1.1]heptane-6-carboxylate

A mixture of4-(6-fluoropyridin-3-yl)-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridine-3-carbonitrile(Intermediate B3; 1.70 g, 8.55 mmol),3,6-diaza-bicyclo[3.1.1]heptane-6-carboxylic acid tert-butyl ester (1.70g, 8.55 mmol) and K₂CO_(3(s)) (7.88 g, 57.0 mmol) in DMSO (7 mL) wasstirred 12 h at 90° C. The resultant thick slurry was diluted withadditional DMSO (2 mL) and stirred for 12 h at 90° C. The mixture wascooled to ambient temperature and diluted with water (100 mL). Theaqueous mixture was washed with DCM. The combined organic extracts weredried over anhydrous MgSO_(4(s)), filtered and concentrated in vacuo.The crude residue was purified by silica chromatography (30-80%EtOAc/hexanes as the gradient eluent system) to cleanly provide thetitle compound (2.87 g, 100% yield). MS (apci) m/z=505.2 (M+H).

Step 2: Preparation of4-(6-(3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridine-3-carbonitriledihydrochloride

A solution oftert-butyl-3-(5-(3-cyano-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridin-4-yl)pyridin-2-yl)-3,6-diazabicyclo[3.1.1]heptane-6-carboxylate(see step 1; 3.05 g, 6.04 mmol) in DCM (20 mL) was treated with 4 N HClin dioxanes (15.1 mL, 60.4 mmol). The resulting mixture was stirred for12 hours at ambient temperature, and then concentrated in vacuo. Thecrude residue was diluted with DCM and toluene, and then sonicatedbefore concentrating in vacuo to afford the title compound as thedihydrochloride salt (2.44 g, quantitative yield). MS (apci) m/z=405.2(M+H).

Compound of Formula II

6-(2-hydroxy-2-methylpropoxy)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile

To a mixture of4-(6-(3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridine-3-carbonitriledihydrochloride (Intermediate B4, 6.0 g, 12.6 mmol) in DCM (30 mL) wasadded triethylamine (5.3 mL, 37.7 mmol), followed by6-methoxynicotinaldehyde (2.59 g, 18.9 mmol) and then sodiumtriacetoxyborohydride (5.33 g, 25.1 mmol) in one portion while stirring.Additional DCM (30 mL) was added and the reaction was stirred at ambienttemperature overnight. The reaction mixture was poured into water (200mL) and extracted with DCM (200 mL). After phase-separation, the organiclayer was washed with water (2×200 mL). The combined aq washes wasback-extracted with DCM (200 mL). The combined organic extracts waswashed with brine (250 mL), passed through a Phase Separator frit andtreated with activated charcoal (Darco G-60, 6 g). After stirred atambient temperature for 2 h, the mixture was vacuum-filtered, rinsedwith DCM (3×10 mL). The filtrate was treated with3-(trimethoxysilyl)propane-1-thiol (11 g, 7.4 mmol) and magneticallystirred overnight. The mixture was vacuum-filtered and concentrated onrotovap to ca. 200 mL. Heptane (150 mL) was added portion-wise to theabove DCM solution while stirring until cloudy. After 90 min stirring,the white slurry was vacuum-filtered, rinsed with heptane (200 mL),yielding the title product as fine crystalline white powder (3.9 g,59%).

Recrystallization of the compound of Formula II in DMSO/water wasperformed as follows. To a reaction flask was charged the compound ofFormula II (10.1 g) and DMSO (110 mL). The mixture was heated to 50° C.until all of the solid was in solution. The mixture was cooled to 25° C.and polish filtered. DMSO (10 mL) was charged through the filter as awash. The resulting solution was heated to 45° C. and water (5 mL) wasslowly added. The mixture was stirred for 30 minutes and a seed bedformed. Water (25 mL) was added over 1 h and the slurry was aged at 45°C. for an additional 1 hour. The slurry was then allowed to cool to 25°C. and stir for 2 hours. The slurry was filtered and the cake was washedwith water (20 mL×3), MeOH (20 mL×2) and MTBE (20 mL×2). The cake wasdried at room temperature in a vacuum oven to give 9.35 g (74%) of thetitle compound. MS (apci) m/z=526.2 (M+H). ¹H NMR (400 MHz, DMSO-d₆) δ:8.64 (d, 1H, J=2.3 Hz), 8.55 (s, 1H), 8.38 (d, 1H, J=2.3 Hz), 8.04 (d,1H, J=2.3 Hz), 7.80 (dd, 1H, J=8.6, 2.3 Hz), 7.64 (dd, 1H, J=8.6, 2.3Hz), 7.27 (d, 1H, J=2.0 Hz), 6.76 (d, 1H, J=8.6 Hz), 6.73 (d, 1H, J=8.2Hz), 4.67 (s, 1H), 3.85 (s, 2H), 3.79 (s, 3H), 3.72 (d, 2H, J=12.5 Hz),3.64 (d, 2H, J=5.9 Hz), 3.51 (br d, 2H), 3.47 (s, 2H), 2.47 (m, 1H),1.55 (d, 1H), 1.20 (s, 6H).

Compound of Formula III

6-(2-hydroxy-2-methylpropoxy)-4-(6-(6-(6-methoxynicotinoyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile

To a mixture of4-(6-(3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridine-3-carbonitriledihydrochloride (Intermediate B4, 4.50 g, 9.43 mmol) in DMSO (63 mL) wasadded 6-methoxynicotinic acid (1.73 g, 11.3 mmol) followed by Hunig'sbase (5.25 mL, 30.2 mmol) and HATU (4.30 g, 11.3 mmol). This was stirredat room temperature for 1 h before it was poured into a mixture of water(500 mL) and sat. NaHCO₃(50 mL), then stirred for another 4 h. Thesuspension was vacuum-filtered, and the solid collected was firsttriturated with MTBE (100 mL), then treated with a flash silicachromatography (1-15% MeOH in DCM with 1% NH₄OH) to yield the titleproduct. This product was recrystallized by dissolving in minimal amountof DCM, followed by slow addition of MTBE until a suspension appeared.Filtration followed by drying under high vac at 40-45° C. for 2 dyielded the title product as crystalline white powder (4.0 g, 78.6%).

Further recrystallization of the title product in CH₃CN/water wasperformed as follows. A mixture of the title product (37 g) in CH₃CN(222 mL) was heated to reflux to obtain a clear solution. Water (333 mL)was then slowly added at the same temperature while stirring. Afteraddition the heating was stopped and the mixture was allowed to cool toRT and stirred overnight. The solid was collected via vacuum-filtrationand drying under high vacuum at 40-45° C. overnight, yielding the titleproduct as crystalline white powder (24 g, 65%). ¹H NMR (DMSO-d⁶) δ8.60-8.65 (d, 1H), 8.53 (s, 1H), 8.49-8.51 (m, 1H), 8.28-8.31 (d, 1H),7.91-7.95 (m, 1H), 7.73-7.78 (m, 1H), 7.23-7.25 (m, 1H), 6.81-6.85 (m,1H), 6.65-6.69 (d, 1H), 4.84-4.94 (br.m, 1H), 4.66 (s, 1H), 4.51-4.63(br.m, 1H), 4.04-4.20 (br.m, 1H), 3.88 (s, 3H), 3.83 (s, 2H), 3.60-3.63(m, 2H), 3.42-3.53 (br.m, 1H), 2.75-2.85 (m, 1H), 1.63-1.69 (m, 1H),1.18 (s, 6H). MS (apci) m/z=540.2 (M+H).

Compound of Formula IV

6-(2-hydroxy-2-methylpropoxy)-4-(6-(4-hydroxy-4-(pyridin-2-ylmethyl)piperidin-1-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile

To a suspension of4-(6-fluoropyridin-3-yl)-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridine-3-carbonitrile(Intermediate B3, 90 mg, 0.276 mmol) in DMSO (2 mL) was added DIEA (193μL, 1.10 mmol), followed by the addition of4-(pyridin-2-ylmethyl)piperidin-4-ol hydrochloride (69 mg, 0.303 mmol).The reaction mixture was stirred at 90° C. for 60 h, then purifieddirectly by C18 reverse phase chromatography (using 5-95% acetonitrilein water with 0.1% TFA as the gradient eluent). Fractions containing thedesired product were combined, partially concentrated in vacuo to removethe ACN, then partitioned between saturated NaHCO₃(aq) and DCM. Thebiphasic mixture was extracted with additional DCM (2×). The combinedorganic extracts were dried over anhydrous MgSO₄(s), filtered andconcentrated in vacuo. The residue was sonicated in Et₂O (2 mL) and thenconcentrated in vacuo to afford the title compound (48 mg, 35% yield).MS (apci) m/z=499.2 (M+H).

Further recrystallization and isolation of Form A: To a reaction vesselwas added the compound of Formula IV (10 g), DMSO was added (50 mL) andthe mixture was stirred. After 10 minutes, all the compound of FormulaIV was dissolved and then water (4×5 mL, 20 mL total) was addeddropwise. After stirring for 10 minutes a thin suspension formed.Additional water (3×5 mL, 15 mL total) was added and after stirring for30-60 minutes the suspension was filtered. The cake was washed withwater (10 mL), MTBE (50 mL) and then the solid was dried at ambienttemperature under vacuum to yield 9.2 g of the compound of Formula IV.The crystalline solid was analyzed by DSC and XRPD producing aconsistent endotherm and diffraction pattern of Form A for the compoundof Formula IV.

Example 3: Polymorph Salt Screens for the Compound of Formula I

A. Solubility Studies of the Freebase of the Compound of Formula I

The solubility of the free base of the compound of Formula I wasexamined in 13 solvents at 25° C. and 60° C. The solubility was alsodetermined in mixed solvent systems such as DCM/EtOH, DMSO/EtOH,DMSO/water, THF/EtOH, and THF/water.

The solubility studies were conducted as follows. 10-20 mg of the freebase was stirred in 1 mL of solvent overnight at room temperature; thesolubility at 25° C. was noted. The samples were then heated to 60° C.Should the solids dissolve on heating, more freebase was added in 3 to 5mg portions. The results of the solubility test are shown in Tables 5-10and FIGS. 1A-1E.

TABLE 5 General solubility of the freebase of the compound of formula ISolubility at 25° C. Solvent (mg/mL) Solids dissolve at 60° C.? EtOAc0.36 No iPAc 0.25 No MeOH 0.07 No EtOH 0.04 No toluene 0.35 No acetone0.44 No IPA 0 No MEK 0.69 No THF 2.59 Yes (between 10.2-13.0 mg/mL) ACN0.33 No DMA 12.9 >40 mg/mL DCE 4.6 Not studied DCM 15.5 N/A water 0.07N/A 0.1M HCl 1.5 Not studied

The freebase exhibited solubilities of 2.59 mg/mL, 12.9 mg/mL, and 15.5mg/mL in THF, DMA, and DCM, respectively. For most of the solventsystems examined, the freebase did not dissolve after heating to 60° C.10-13 mg of freebase dissolved in THF at 60° C., although the solutionturned milky at 13 mg. Solubility study in DMA at 60° C. was stoppedafter a total of 40 mg of freebase was added.

TABLE 6 The solubility of the freebase of the compound of Formula I inDCM/EtOH at room temperature % DCM in EtOH Solubility (mg/mL) 0 0.05 100.06 20 0.26 30 0.87 40 2.53 50 7.42 60 16.6 70 22.7 80 29.0 90 34.2 10017.3

The solubility of the freebase in DCM/EtOH mixtures is shown in FIG. 1Aand Table 6, above.

TABLE 7 The solubility of the freebase of the compound of Formula I inDMSO/EtOH at room temperature % DMSO in EtOH Solubility (mg/mL) 0 0.0510 0.09 20 0.18 30 0.39 40 0.61 50 1.14 60 1.80 70 2.57 80 4.28 90 4.44100 6.96

The solubility of the freebase in DMSO/EtOH mixtures is shown in FIG. 1Band Table 7, above.

TABLE 8 The solubility of the freebase of the compound of Formula I inDMSO/H₂O at room temperature % DMSO in H₂O Solubility (mg/mL) 0 0 100.09 20 0.08 30 0.09 40 0.10 50 0.09 60 0.10 70 0.15 80 0.35 90 1.52 1006.96

The solubility of the freebase in DMSO/H₂O mixtures is shown in FIG. 1Cand Table 8, above.

TABLE 9 The solubility of the freebase of the compound of Formula I inTHF/EtOH at room temperature % THF in EtOH Solubility (mg/mL) 10 0.29 200.34 30 0.55 40 0.82 50 1.38 60 1.97 70 2.51 80 3.09 90 3.18 100 2.59

The solubility of the freebase in THF/EtOH mixtures is shown in FIG. 1Dand Table 9, above.

TABLE 10 The solubility of the freebase of the compound of Formula I inTHF/H₂O at room temperature % THF in EtOH Solubility (mg/mL) 10 0.15 200.13 30 0.35 40 1.36 50 2.50 60 3.76 70 6.17 80 6.65 90 6.56 100 2.59

The solubility of the freebase in THF/H₂O mixtures is shown in FIG. 1Eand Table 10, above.

Studies were undertaken to determine possible recrystallizationconditions of the freebase of the compound of Formula I as shown inTable 11.

TABLE 11 Recrystallization attempts on the freebase of the compound ofFormula I Scale Solvent (vol)// Temp Time LTF (g) Antisolvent (vol) (°C.) (hr) (mg/mL) Notes 0.05 DMSO (18)// 70° C. 16 1.20 Freebasedissolved in DMSO at 70° C. EtOH 200 proof to RT Slow addition of EtOH.Solids slowly (27) appear 1 hr after cooling started. Cooled overnight.Filtered. 78.6% recovered 0.10 DMSO (18)// 70° C. 16 1.20 Freebasedissolved in DMSO at 70° C. EtOH 200 proof to RT Slow addition of EtOHuntil hazy. Solids (25) appear quickly at 70° C. Cooled overnight.Filtered. 83.6% recovered. 0.10 DMSO (18)// 70° C. 16 2.26 Freebasedissolved in DMSO at 70° C. EtOH 200 proof to RT Slow addition of EtOHuntil hazy. Cooled (18) overnight. Filtered. 80.5% recovered. 5.0 DMSO(20)// 70° C. 16 1.6 Freebase dissolved in DMSO at 70° C. EtOH 200 proofto RT Slow addition of EtOH until hazy. Cooled (20) overnight. Filtered88% recovered. 0.12 DMSO 70° C. 16 1.00 Freebase dissolved in DMSO at70° C. (18)//EtOH 200 to RT Slow addition of EtOH until hazy. Cooledproof (33) overnight. Filtered. 85% recovered. 0.10 DMSO 70° C. 16 1.03Freebase dissolved in DMSO at 70° C. (18)//EtOH 200 to RT Cooled to 40°C., becomes hazy, slow proof (28) addition of EtOH. Solids appearimmediately. 78.2% recovered. 0.10 DMSO 70° C. 72 0.70 Freebasedissolved in DMSO at 70° C. (18)//EtOH 190 to RT Slow addition of EtOHuntil hazy. Cooled proof (28) overnight. Not filtered. 0.10 90% DCM/EtOH40° C. 16 2.38 Freebase dissolved in 90% DCM in EtOH (25)// EtOH to RTat 40° C. Slow addition of EtOH until hazy. 200 proof (19) Solids crashout semi-slowly at 40° C. Cooled overnight. 0.10 66% DCM/EtOH 40° C. 163.70 Freebase dissolved in 66% DCM in EtOH (30)// EtOH to RT at 40° C.Slow addition of EtOH, still 200 proof (20) solution. Cooled overnight.Solids present in the morning 1.0 DMSO 100° C. 24 1.1 Freebase dissolvedin DMSO at 100° C. (6)//H2O(24) to RT Slow addition of H₂O to makeslurry. Heated for 10 hrs at 100° C., then cooled overnight to RT. Washwith 20 volumes of H₂O. Dried in oven. Recovery - TBD 1.0 DMSO 100° C.24 0.9 Freebase dissolved in DMSO at 100° C. (10)//EtOH 200 to RT Cooledto 75° C. Slow addition of EtOH to proof (20) make slurry. Stir for 10hrs at 75° C., then cooled overnight to RT. Filtered. Wash with 20volumes of H₂O. Dried in oven. Recovery - TBD 1.0 EtOH 200 proof 75° C.24 0.6 Freebase slurried in EtOH at 75° C. Heat (20) to RT for 24 hrs at75° C. Cooled to RT and filtered. Wash with 20 volumes of H₂O. Dried inoven. Recovery - TBD

The free base of the compound of Formula I was found to dissolve in 18volumes DMSO at 70° C. and in 6 volumes DMSO at 100° C. EtOH was addedto a DMSO solution at 70° C.; the solution became cloudy after ˜18volumes of EtOH (1:1 DMSO/EtOH). Additional EtOH forced solids to crashout at 70° C.

B. Characterization of the Freebase of the Compound of Formula I

The compound of Formula I, prepared as described in Example 1, above,was analyzed by PLM, XRPD, DSC, TGA, DVS, FTIR, and ¹H NMR and wasidentified as Form A (FIGS. 2A-2G). The X-ray powder diffraction scan isshown in FIG. 2A. The peaks of the X-ray powder diffraction scan isshown in Table 12, below. An overlay of X-ray powder diffraction scansof different lots of the freebase of the compound of Formula I is shownin FIG. 2B. A small endothermic event was observed by DSC from an onsetof around 187° C. followed by an endothermic event observed from anonset of around 224° C. (FIG. 2C). The isothermic (25° C.) DVS scan isshown in FIG. 2D. TGA analysis indicated a weight loss of 1.1% from theonset of heating to around 238° C. (FIG. 2E). The FTIR spectrum is shownin FIG. 2F. The NMR (in DMSO-d⁶) spectrum is shown in FIG. 2G.

TABLE 12 XRPD peaks of Form A of the compound of Formula I 2-Theta d(Å)Height H % 4.44 19.90 92.00 100.00 9.05 9.76 5.70 6.20 11.29 7.83 1.601.70 13.48 6.56 32.30 35.10 14.58 6.07 73.80 80.20 14.94 5.93 8.50 9.3015.29 5.79 0.40 0.40 15.66 5.65 2.90 3.10 16.41 5.40 5.10 5.60 17.195.15 9.90 10.80 17.45 5.08 16.70 18.10 17.75 4.99 12.90 14.10 18.27 4.8536.40 39.60 18.77 4.73 36.10 39.30 19.69 4.50 4.00 4.40 20.03 4.43 13.7014.90 20.51 4.33 4.90 5.40 20.95 4.24 24.00 26.10 21.14 4.20 6.30 6.9021.53 4.12 8.00 8.70 21.92 4.05 14.10 15.30 22.25 3.99 4.90 5.30 22.453.96 15.60 17.00 23.39 3.80 1.30 1.40 23.80 3.73 8.00 8.70 24.12 3.695.20 5.60 24.57 3.62 30.30 33.00 24.94 3.57 1.80 2.00 25.32 3.51 5.005.40 25.60 3.48 2.50 2.70 26.45 3.37 8.00 8.70 26.65 3.34 8.90 9.6027.17 3.28 2.50 2.70 27.72 3.22 15.70 17.10 28.25 3.16 1.30 1.40 28.563.12 3.80 4.10 29.31 3.04 1.60 1.70 29.64 3.01 3.40 3.70 30.16 2.96 0.800.80 30.75 2.91 3.50 3.80 31.24 2.86 2.00 2.20 31.72 2.82 1.00 1.1032.19 2.78 0.50 0.60 33.16 2.70 1.30 1.40 34.05 2.63 2.30 2.50 34.402.60 1.10 1.20 35.02 2.56 0.80 0.90 35.44 2.53 1.40 1.50

C. Initial Salt Screen

An initial salt screen was conducted on the compound of Formula I usinginorganic and organic acid counterions and DCM or DMA as solvent(solubility of the freebase of the compound of Formula I in DCM=15.5mg/mL; solubility of the freebase of the compound of Formula I inDMA=12.9 mg/mL). The combinations of acid and solvent are shown in Table13, below.

TABLE 13 Results from the primary salt screen Appearance After Afterheating After heating to to 40° C. and dilution 40° C. for cooling overAfter with Acid Solvent Initial³ After 13 h 4.5 h night evaporation MTBEMaleic Acid¹ DCM Solid, pink Solid, N/A N/A N/A N/A tint slight pinktint L-Lactic Acid DCM Clear, no Clear, no N/A N/A Brown, N/A solidsolid, pink sticky oil at tint dryness Acetic Acid DCM Clear, no Clear,no N/A N/A Beige solid N/A solid solid, at dryness yellow tint AdipicAcid DCM Slightly Slightly Slightly Slightly N/A N/A cloudy, cloudy,cloudy, cloudy, fine few hard to tell hard to tell particles “solids” ifsolids if solids floating in on wall of present present solution vialFumaric Acid DCM Cloudy Cloudy, Cloudy, Cloudy, fine N/A N/A fine solidfine solid solid D-Glutamic DCM Cloudy Slightly Cloudy, Cloudy, N/A N/AAcid cloudy, milky milky hard to tell if solids present D-Malic Acid¹DCM Solids, Solids, N/A N/A N/A N/A slight pink slight pink color colorL-Malic Acid¹ DCM Solids, Solids, N/A N/A N/A N/A white white (pinktint) MsOH DCM Solids, Clear, Clear, Clear, milk- N/A N/A white brownmilky-like like solids on solids on solid on walls walls walls (lostsolvent cap loose) pTsOH•H₂O DCM Cloudy, Cloudy, Cloudy, Cloudy, N/A N/Amilky milky milky milky Benzenesulfonic DCM Clear, Clear, Some Somesolids N/A N/A Acid light light pink, solids on on walls, yellow tintfew solids walls, milky milky solution solution Benzoic Acid DCM Clear,Clear, N/A N/A Beige solid N/A light light at dryness yellow tint yellowMalonic Acid DCM Clear, White, Some Some solids N/A N/A light cloudy,solids on on walls, fine yellow tint few solids walls, fine solids onwalls of solid vial HBr/H₂O DCM Cloudy, Cloudy, Cloudy, Cloudy, N/A N/Apink milky, milky milky, pink “sticky” pink pink solids solids on wallsolid on “sticky” on wall of of vial walls of solids on vial vial wallof vial Succinic Acid DCM Clear, Very fine Fine Very fine N/A N/A lightparticles particles, particles yellow tint some solid on wallsL-Tartaric Acid² DCM Cloudy, Cloudy, Solids, Solids, pink N/A N/A smallcan't tell if pink tint tint particles solids are filterable HCl/H₂O DCMCloudy Cloudy, Brown Brown N/A N/A milky “sticky” “sticky” solids onsolids on wall, milky wall, milky Sulfuric Acid DCM Clear, Clear, Solidon Clear N/A N/A some solid some solid sides of solution, stuck to stuckto vial, some brown/white walls of walls of solid “solids” on vial vialfloating wall of vial Phosphoric Acid DCM Clear, Clear, Clear Clearyellow N/A N/A yellow tint yellow tint yellow solution, solution, brownsolids brown stuck to vial solids stuck to vial Citric Acid² DCM Cloudy,Solids, N/A N/A N/A N/A small white particles Propionic Acid DCM ClearClear, N/A N/A Beige solid N/A yellow tint at dryness D-Tartaric Acid²DCM Cloudy, Solids, N/A N/A N/A N/A some white solids L-Glutamic DCMSolids Slightly Slightly Cloudy, N/A N/A Acid floating in cloudy,cloudy, milky solution hard to tell hard to tell if solids if solidspresent present Maleic Acid DMA Clear, Clear, Clear, light Clear, lightN/A Cloudy light light yellow tint yellow tint emulsion yellow tintyellow tint L-Lactic Acid DMA Clear, Clear, Clear, light Clear, lightN/A Cloudy light light yellow tint yellow tint emulsion yellow tintyellow tint Acetic Acid DMA Clear, Clear, Clear, light Clear, light N/ACloudy light light yellow tint yellow tint emulsion yellow tint yellowtint Adipic Acid DMA Clear, Clear, Clear, light Clear, light N/A Cloudylight light yellow tint yellow tint emulsion yellow tint yellow tintFumaric Acid DMA Clear, Clear, Clear, light Clear, light N/A Cloudylight light yellow tint yellow tint emulsion yellow tint yellow tintD-Glutamic DMA Cloudy Cloudy Cloudy Cloudy N/A Cloudy Acid emulsionemulsion emulsion emulsion D-Malic Acid DMA Clear, Clear, Clear, lightClear, light N/A Cloudy light light yellow tint yellow tint emulsionyellow tint yellow tint L-Malic Acid DMA Clear, Clear, Clear, lightClear, light N/A Cloudy light light yellow tint yellow tint emulsionyellow tint yellow tint Methanesulfonic DMA Clear, Clear, Clear, lightClear, light N/A Cloudy Acid light light yellow tint yellow tintemulsion yellow tint yellow tint pTsOH•H₂O DMA Clear, Clear, Clear,light Clear, light N/A Cloudy light light yellow tint yellow tintemulsion yellow tint yellow tint Benzene DMA Clear, Clear, Clear, lightClear, light N/A Cloudy sulfonic Acid light light yellow tint yellowtint emulsion yellow tint yellow tint Benzoic Acid DMA Clear, Clear,Clear, light Clear, light N/A Cloudy light light yellow tint yellow tintemulsion yellow tint yellow tint Malonic Acid DMA Clear, Clear, Clear,light Clear, light N/A Cloudy light light yellow tint yellow tintemulsion yellow tint yellow tint HBr/H₂O DMA Solids Filtered N/A N/A N/AN/A Off white white Solids - Slow filtration Succinic Acid DMA Clear,Clear, Clear, light Clear, light N/A Cloudy light light yellow tintyellow tint emulsion yellow tint yellow tint L-Tartaric Acid3 DMA Clear,Clear, Clear, light Clear, light N/A Clear light light yellow tintyellow tint solution yellow tint yellow tint Propionic Acid DMA Clear,Clear, Clear, light Clear, light N/A Cloudy light light yellow tintyellow tint emulsion yellow tint yellow tint Sulfuric Acid DMA Clear,Clear, Clear, light Clear, light N/A Cloudy light light yellow tintyellow tint emulsion yellow tint yellow tint Phosphoric Acid DMA Clear,Clear, Clear, light Clear, light N/A Cloudy light light yellow tintyellow tint emulsion yellow tint yellow tint Citric Acid DMA Clear ClearClear Clear solution N/A Cloudy solution solution solution emulsionHCl/H₂O DMA Solids, Filtered N/A N/A N/A N/A white white Solids,Propionic Acid DMA Clear, Clear, Clear, light Clear, light N/A Cloudylight light yellow tint yellow tint emulsion yellow tint yellow tintD-Tartaric Acid² DMA Clear, Clear, Clear, light Clear, light N/A Cloudylight light yellow tint yellow tint emulsion yellow tint yellow tintL-Glutamic DMA Cloudy Cloudy Cloudy Cloudy N/A Cloudy Acid emulsionemulsion emulsion emulsion emulsion HCl/ DMA Emulsion Emulsion EmulsionEmulsion N/A Emulsion Dioxane ¹Acids that provided solids after 13 hoursthat were able to be filtered. ²Solid was filtered after heating andcooling. ³Observations for DCM experiments were recorded after allexperiments were set-up (~1 hr after first vial).

Samples that provided solids after 13 hours at room temperature werefiltered and analyzed. Samples that did not provide solids afterovernight stirring and were clear solutions were evaporated to dryness(reactions in DCM) or charged with MTBE (reactions in DMA). Theresulting solids were isolated and analyzed. Samples that providedunfilterable solids were heat cycled; solids (if present) were isolatedthrough filtration after heating and cooling. The appearances of solidsare shown in Table 13, above.

Solids formed from the following acids were filtered and analyzed:maleic acid, D-malic acid, L-malic acid, citric acid, L-tartaric acid,D-tartaric acid, HBr, and HCl. For the following acids, solids formedafter evaporation of DCM were also analyzed: acetic acid, benzoic acid,and propionic acid.

This initial screen provided eleven salts for further analysis (PLM,DSC, etc.). The salts were formed from the following acids: maleic acid,acetic acid, D-malic acid, L-malic acid, citric acid, L-tartaric acid,D-tartaric acid, benzoic acid, propionic acid, HCl and HBr.

Additional screenings for sulfuric and phosphoric acid salts were alsoperformed as shown in Table 14, below.

TABLE 14 Screen of conditions for sulfuric and phosphoric acid saltsSolvent Acid Time Scale (Vol) (equivalents) Temperature (h) Notes 20 mgTHF H₂SO₄/H₂O 25° C. 24 Never goes into solution, Milky (100) (1.1)solution overnight, solids stuck to walls of vial 20 mg EtOH H₂SO₄/H₂O25° C. 24 Thick solid forms upon addition of (100) (1.1) acid, turned tosolid gel, Milky solution overnight 20 mg 1:1 H₂SO₄/H₂O 25° C. 24 Uponaddition of acid, most solids DCM/EtOH (1.1) dissolve. Sticky solids onwall. (100) Milky solution overnight 20 mg 4:1 H₂SO₄/H₂O 25° C. 24Freebase fully dissolved before DCM/EtOH (1.1) addition of acid. Acidforms almost (100) immediately. Filterable solids overnight 20 mg THFH₃PO₄/H₂O 25° C. 24 Never goes into solution, initially (100) (1.1)thick solids form. Milky solution overnight 20 mg EtOH H₃PO₄/H₂O 25° C.24 Never goes into solution, thick (100) (1.1) solids form. Milkysolution overnight 20 mg 1:1 H₃PO₄/H₂O 25° C. 24 Sticky solids on wallof vial, DCM/EtOH (1.1) becomes thicker over time. Milky (100) solutionovernight 20 mg 4:1 H₃PO₄/H₂O 25° C. 24 Freebase fully dissolved beforeDCM/EtOH (1.1) addition of acid. Acid forms almost (100) immediately.GEL overnight

The freebase of the compound of formula I was found to dissolve in 4:1DCM/EtOH while exhibiting limited solubility in THF, EtOH, and 1:1DCM/EtOH. Conditions listed in Table 14, above, did not provide afilterable phosphoric acid salt. The use of sulfuric acid in 4:1DCM/EtOH provided a filterable solid after stirring overnight.

Solids formed from the acids indicated in Table 15 were analyzed by PLMand DSC; the appearances and melting points of these solids asdetermined by DSC are shown in Table 15. The DSC curves are shown inFIGS. 3-14.

TABLE 15 Appearance and melting point of isolated salts Appearance ofsolid after filtration or solvent Acid evaporation Melting Point (DSC)N/A Freebase 229.23° C. Maleic Acid Grey/tan solid 226.84° C. D-MalicAcid Grey/tan solid 212.69° C. L-Malic Acid Grey/tan solid 213.52° C.Citric Acid Grey/tan solid 197.53° C. L-Tartaric Acid Grey/tan solid222.66° C. D-Tartaric Acid Grey/tan solid 219.24° C. Acetic Acid Beigesolid 229.06° C. Benzoic Acid Beige solid 229.16° C. Propionic AcidBeige solid 228.77° C. HBr Beige solid 225.25° C. HCl Off White solid241.25° C. H₂SO4 Off White solid 285.95° C.

The DSC thermogram for the maleic acid salt identified during theinitial salt screening is shown in FIG. 3. An endothermic event wasobserved by DSC from an onset of around 224° C. A small endothermicevent was observed by DSC from an onset of around 208° C.

The DSC thermogram for the acetic acid salt identified during theinitial salt screening is shown in FIG. 4. An endothermic event wasobserved by DSC from an onset of around 227° C.

The DSC thermogram for the D-malic acid salt identified during theinitial salt screening is shown in FIG. 5. An endothermic event wasobserved by DSC from an onset of around 211° C.

The DSC thermogram for the benzoic acid salt identified during theinitial salt screening is shown in FIG. 6. An endothermic event wasobserved by DSC from an onset of around 228° C.

The DSC thermogram for the L-tartaric acid salt identified during theinitial salt screening is shown in FIG. 7. An endothermic event wasobserved by DSC from an onset of around 216° C.

The DSC thermogram for the citric acid salt identified during theinitial salt screening is shown in FIG. 8. An endothermic event wasobserved by DSC from an onset of around 190° C.

The DSC thermogram for the propionic acid salt identified during theinitial salt screening are shown in FIG. 9. An endothermic event wasobserved by DSC from an onset of around 226° C. An endothermic event wasobserved by DSC from an onset of around 148° C.

The DSC thermogram for the D-tartaric acid salt identified during theinitial salt screening is shown in FIG. 10. An endothermic event wasobserved by DSC from an onset of around 210° C. A small endothermicevent was observed by DSC from an onset of around 159° C.

The DSC thermogram for the L-malic acid salt identified during theinitial salt screening is shown in FIG. 11. An endothermic event wasobserved by DSC from an onset of around 211° C.

The DSC thermogram for the sulfuric acid salt identified during theinitial salt screening are shown in FIG. 12. An endothermic event wasobserved by DSC from an onset of around 277° C. A small endothermicevent was observed by DSC from an onset of around 35° C.

C. Scale Up and Optimization of Selected Salts

The hydrochloric acid (HCl) and hydrobromic acid (HBr) salts preparedduring the primary salt screen was scaled-up as shown in Table 16 forfurther analysis.

TABLE 16 Scale up of HCl and HBr salts Volume of Volume Max TempAntisolvent Isolated Scale (g) Acid (equiv) DMA (° C.) (MTBE) Ageingtime solids 0.2 g 36% HCl 75 28° C. 125 Overnight 152 mg, (1.1) withstirring 71% Yield 6.2% Cl- found (6.5% Expected) 0.2 g 48% HBr 50 40°C. 150 Overnight 215 mg (1.1) without 86% Yield stirring 12% Br- Found(13.5% Expected

The DSC thermogram for the hydrochloric acid salt from ˜200 mg scalereaction is shown in FIG. 13. An endothermic event was observed by DSCfrom an onset of around 233° C.

The DSC thermogram for the hydrobromic acid salt from ˜200 mg scalereactions is shown in FIG. 14. An endothermic event was observed by DSCfrom an onset of around 195° C.

Alternative conditions for the formation of the HCl salt were examined.For example, the volume of DMA was decreased to 50 volumes and thereaction mixture was heated to 40° C. as shown in Table 17 (entry 1).HCl was added at this temperature; the resulting mixture was then cooledto room temperature. The DSC thermogram for hydrochloric acid saltprepared this way is shown in FIG. 15A. An endothermic event wasobserved by DSC from an onset of around 230° C.

The HCl salt was also prepared using a mixed solvent system of 1:1DCM/EtOH in 50 vol at 30° C. The DSC thermogram of hydrocholoric acidsalt prepared this way is shown in FIG. 15B. An endothermic event wasobserved by DSC from an onset of around 224° C.

TABLE 17 Alternative conditions for the preparation of HCl salt Volumeof Acid Volume Max Temp Antisolvent Ageing Isolated Scale (g) (equiv.)DMA (° C.) (MTBE/Vol) time solids 0.2 g 36% HCl 75 28° C. 125 overnight152 mg, (1.1) with stirring 71% Yield 6.2% Cl- found (6.5% Expected) 0.2g 48% HBr 50 40° C. 150 overnight 215 mg (1.1) without 86% Yieldstirring 12% Br- Found (13.5% Expected

Four salts (HCl, HBr, L- and D-malic acid salts) and the freebase of thecompound of formula I were scaled up to 2 grams (Table 18). The foursalts were analyzed and characterized by PLM, DSC, TGA, DVS, XRPD, FTIR,and ¹H NMR (FIGS. 16A-16F, 17A-17D, and 18A-18H).

TABLE 18 Scale-up of HCl, HBr, L-Malic and D-Malic salts Scale SolventTime Melting Isolated (g) (Vol) Acid (eq) (h) Temp Point (DSC) yield (%)Notes 2.0 DMA (50) HCl (1.1)  3 hrs 40° C. to 240.61° C. 1.88 g HCladded at RT (87.8%) 40° C. Filtered. White solid 2.0 3.6:1 HCl (1.1) 20hr 25° C. 237.99° C. 1.93 g Chlorine analysis DCM/EtOH (90.1%) shows(46) 1 eq. HCl 2.0 3.6:1 HBr (1.1) 20 hr 25° C. 237.91° C. 2.26 gBromine analysis DCM/EtOH (97.4%) shows (46) 1 eq. HBr 2.0 3.6:1 L-Malic20 hr 25° C. 208.31° C. 1.64 g DCM/EtOH acid 66.8% (46) (0.97) 2.0 3.6:1D-Malic 20 hr 25° C. 208.25° C. 1.60 g DCM/EtOH acid 65.2% (46) (0.97)2.2 3.6:1 L-Malic 20 hr 25° C. N/A 2.11 g DCM/EtOH acid 75.9% (46)(0.97) 2.5 3.6:1 D-Malic 20 hr 25° C. N/A 2.41 g DCM/EtOH acid 76.2%(46) (0.97)

Chlorine and bromine analysis verified the formation of thecorresponding mono-HX salts. The L- and D-malic acid salts were providedin 75-78% yield; the HCl salt was prepared in 90% yield.

For the HCl salt (DMA), an endothermic event was observed by DSC from anonset of around 233° C. (FIG. 16A); a small exothermic event wasobserved by DSC from an onset of around 253° C. For the HCl salt(EtOH/DCM), an endothermic event was observed by DSC from an onset ofaround 223° C.; a small endothermic event was observed by DSC from theonset of around 71° C. (FIG. 16D). Isothermic (25° C.) DVS analysisindicated that the HCl salt (DMA) is hygroscopic (FIG. 16B). A mass gainof approximately 5% was observed at 80% relative humidity. Somehysteresis was observed. Upon removal from the chamber, the sample ofHCl salt remained a free-flowing powder; no observable deliquescence wasnoted. TGA analysis of the HCl salt (DMA) indicated a weight loss of7.4% from the onset of heating to around 255° C. (FIG. 16C). TGAanalysis of the HCl salt (EtOH/DCM mixed solvent system) indicated aweight loss of 8.2% from the onset of heating to around 255° C. (FIG.16D). An overlay of the X-ray powder diffraction scans of HCl salt(prepared in DMA, from different batches, before/after DSV) and HCl salt(EtOH/DCM) is shown in FIG. 16E. ¹H NMR spectrum of HCl salt (preparedin EtOH/DCM) in DMSO-d₆ is shown in FIG. 16F.

For the HBr salt, an endothermic event was observed by DSC from an onsetof around 215° C.; a small endothermic event was observed by DSC from anonset of around 34° C. (FIG. 17A). TGA analysis of the HBr saltindicated a weight loss of approximately 10.3% from the onset of heatingto around 255° C. (FIG. 17A). An X-ray powder diffraction scan of theHBr salt is shown in FIG. 17B. FTIR spectrum of the HBr salt is shown inFIG. 17C. ¹H NMR spectrum of the HBr salt in DMSO-d₆ is shown in FIG.17D.

For the L-malic acid salt, an endothermic event from an onset of around205° C. was observed by DSC (FIG. 18A). For the D-malic acid salt, anendothermic event from an onset of around 206° C. was observed by DSC(FIG. 18B). TGA analysis of the L-malic acid salt indicated a weightloss of approximately 17.7% from the onset of heating to around 253° C.(FIG. 18A). TGA analysis of the D-malic acid salt indicated a weightloss of approximately 18.4% from the onset of heating to around 250° C.(FIG. 18B). Isothermic (25° C.) DVS analysis indicated that both L-malicacid and D-malic acid salts are hygroscopic (FIGS. 18C and 18D). A massgain of approximately 2.8% was observed at 80% RH. Some hysteresis wasobserved. Upon removal from the chamber, the samples of L-malic acid andD-malic acid salts each remained a free-flowing powder; no observabledeliquescence was noted. An overlay of X-ray powder diffraction scans ofthe L- and D-malic acid salts is shown in FIG. 18E. An overlay of FTIRspectra of the L- and D-malic acid salts is shown in FIG. 18F. ¹H NMRspectra of the L- and D-malic salts in DMSO-d⁶ are shown in FIG.18G-18H, respectively.

Two batches of the L-malic acid salt were subjected to DSC, TGA, andXRPD analysis. For the L-malic acid salt (batch A), an endothermic eventfrom an onset of around 210° C. was observed by DSC (FIG. 18I). TGAanalysis of the L-malic acid salt (batch A) indicated a weight loss ofapproximately 18.0% from the onset of heating to around 250° C. (FIG.18J). For the L-malic acid salt (batch B), an endothermic event from anonset of around 217° C. was observed by DSC (FIG. 18K). TGA analysis ofthe L-malic acid salt (batch B) indicated a weight loss of approximately17.7% from the onset of heating to around 250° C. (FIG. 18L). An overlayof X-ray powder diffraction scans of the L-malic acid salt (batches Aand B) and the free base of the compound of Formula I is shown in FIG.18M.

The solubility of the HCl, HBr, L-malic acid, and D-malic acid salts ofthe compound of Formula I in different solvent systems was studied.

The solubility test of the HCl salt was conducted as follows.Approximately 10-20 mg of the HCl salt was stirred in 1 mL solvent. Ineach solvent system, some solids were observed after two hours. Thesamples were subsequently heated to 65° C. for 3 hours when theappearance was noted. The resulting mixture was then allowed to cool toroom temperature overnight. The results of the solubility test are shownin Table 19.

TABLE 19 Solubility of the HCl salt Appearance at Solids dissolveSolubility at Solvent 25° C. after 2 h at 65° C.? 25° C. (mg/mL) WaterSolids No 0.24 MeOH Solids No 0.58 EtOH Solids No 0.12 IPA Solids No 0EtOAc Solids No 0 iPAc Solids No 0 THF Solids No 0 ACN Solids No 0Toluene Solids No 0 DCM Solids N/A 0.10 Acetone Solids N/A 0 MEK SolidsNo 0 MTBE Solids No 0 2-methyl THF Solids No 0 DCE Solids No 0 DMSOSolids Close to 5.94 18.7 mg/mL 0.1M HCl Solids Not studied 2.18

The HCl salt was found to be insoluble in IPA, EtOAc, iPAc, THF, ACN,toluene, acetone, MEK, MTBE, 2-methyl THF, and DCE. A solubility of <1mg/mL was observed in water, MeOH, EtOH, and DCM. A solubility ofapproximately 6 mg/mL was observed in DMSO.

The solubility test of the HBr salt was conducted as follows.Approximately 10-20 mg of the HBr salt was stirred in 1 mL solvent. Ineach solvent system, some solids were observed after two hours. Thesamples were subsequently heated to 65° C. for 3 hours when theappearance was noted. The resulting mixture was then allowed to cool toroom temperature overnight. The results of the solubility test are shownin Table 20.

TABLE 20 Solubility of the HBr salt Appearance at Solids dissolveSolubility at Solvent 25° C. after 2 h at 65° C.? 25° C. (mg/mL) waterSolids No 0 MeOH Solids No 0 EtOH Solids No 0 IPA Solids No 0 EtOAcSolids No 0 iPAc Solids No 0 THF Solids No 0 ACN Solids No 0 DCM SolidsNo 0 Acetone Solids No 0 MEK Solids No 0 MTBE Solids No 0 2-methyl THFSolids No 0 DCE Solids No 0 DMSO Solids No 1.95

The HBr salt exhibited low solubility in the solvent systems examined;the solids did not dissolve upon heating to 65° C. A solubility of 1.95mg/mL was observed in DMSO.

The solubility test of the L-malic acid salt was conducted as follows.Approximately 10-20 mg of the L-malic acid salt was stirred in 1 mLsolvent at room temperature overnight. The results of the solubilitytest are shown in Table 21.

TABLE 21 Solubility of the L-malic acid salt Lot 1: Solubility at 25° C.Lot 2: Solubility at 25° C. Solvent (mg/mL) (mg/mL) water 0.26 0.23 MeOH0.52 0.58 EtOH 0.16 0.15 IPA 0 0 EtOAc 0.20 1.58 iPAc 0 0.33 THF 2.535.42 ACN 0.28 0.85 Toluene 0.29 0.62 DCM 0.24 5.50 Acetone 0.61 1.57 MEK0.51 2.31 MTBE 0 0 2-methyl THF 0.61 1.21 DCE 0.13 4.97 DMSO 14.2 15.40.1M HCl 2.30 Not studied

The solubility test was conducted on two lots of the L-malic acid salt.Some variability in solubility between lots was observed. A solubilityof 14-15 mg/mL was observed in DMSO.

A second solubility study was conducted as follows to determine thesolubility of the L-malic acid salt at elevated temperatures.Approximately 10-20 mg of the L-malate salt was stirred in 1 mL solventat room temperature for 30 min. The samples were heated to 60° C. for3.0 hours; the solubility was noted. The samples were then heated to 70°C. for approximately 3.0 hours; the solubility was noted. The results ofthe second solubility study are shown in Table 22.

TABLE 22 Solubility of the L-malic acid salt at elevated temperaturesSolvent Solids dissolve at 60° C.? Solids dissolve at 70° C.? water NoNo MeOH No N/A EtOH No No IPA No No EtOAc No No iPAc No No THF 65° C.almost 12.3 mg/mL N/A ACN No No Toluene No No DCM N/A N/A Acetone No N/AMEK No No MTBE No No 2-methyl THF No No DCE No No DMSO Yes (>36 mg/mL)  Yes (>56 mg/mL) 0.1M HCl Yes (>13.4 mg/mL) N/A

The solubility test of the D-malic acid salt was conducted as follows.Approximately 10-20 mg of the D-malic acid salt was stirred in 1 mLsolvent overnight. For solubility at elevated temperatures,approximately 10-20 mg of the D-malic acid salt was stirred in 1 mLsolvent at room temperature for 30 min. The samples were heated to 60°C. for 3.0 hours; the solubility was noted. The samples were then heatedto 70° C. for approximately 3.0 hours; the solubility was noted. Theresults of the solubility study are shown in Table 23.

TABLE 23 Solubility of the D-malate salt Solubility at Solids dissolveSolids dissolve Solvent 25° C. (mg/mL) at 60° C.? at 70° C.? water 0.35No No MeOH 0.49 No N/A EtOH 0.14 No No IPA 0 No No EtOAc 0.21 No No iPAc0.12 No No THF 2.65 At 65° C. N/A ~10 mg/mL ACN 0.23 No No Toluene 0 NoNo DCM 0.17 N/A N/A Acetone 0.65 No N/A MEK 0.61 No No MTBE 0 No No2-methyl THF 0.62 No No DCE 0.14 No No DMSO 16.7 Yes (>40 mg/mL) Yes(>56 mg/mL) 0.1M HCl 2.90 Yes (>15 mg/mL) N/A

The preparations of the L-malic acid salt and the HCl salt were scaledup further. The L-malic acid salt was prepared in 77.7% yield and theHCl salt was provided a 91% yield. Conditions for the formation of theL-malic acid salt were then optimized to increase the yield as shown inTable 24.

TABLE 24 HCl salt and L-malic acid salt scale up Scale Solvent Acid TimeIsolated (g) (Vol) (eq.) (hr) Temp yield (%) Notes 10 3.6:1 L-Malic 20hr 25° C. 9.83 g DCM/EtOH acid (77.7%) (46) (0.97) 10 3.6:1 HCl 20 hr25° C. 10.05 g DCM/EtOH (1.1) (91.4%) (46) 2.0 ~3.3:1 L-Malic 20 hr 25°C. 2.01 g The freebase dissolved at RT in 30 vol DCM/EtOH acid (79.4%)4:1 DCM/EtOH. L-Malic acid was (31) (0.97) added as EtOH solution (1vol) 1.5 ~6:1 L-Malic 20 hr 25° C. 1.64 g The freebase dissolved at RTin 30 vol DCM/EtOH acid (86.2%) 4:1 DCM/EtOH. L-Malic acid was (26 vol)(1.05) added as EtOH solution (1 vol) 8.0 ~6:1 L-Malic 20 hr 25° C. 8.70g The freebase dissolved at RT in 25 vol DCM/EtOH acid (86.0%) 9:1DCM/EtOH. L-Malic acid was (26 vol) (1.05) added as EtOH solution (1vol) 8.0 ~6:1 L-Malic 20 hr 25° C. 8.86 g The freebase dissolved at RTin 25 vol DCM/EtOH acid (87.5%) 9:1 DCM/EtOH. L-Malic acid was (26 vol)(1.05) added as EtOH solution (1 vol). The slurry was then cooled to 0°C. for 1.5 hrs before filtration.

The solvent volume was lowered to 30 volumes using a 4:1 ratio ofDCM/EtOH. Addition of L-Malic acid (0.97 equiv) in 1 volume of EtOHbrought the total solvent to 31 volumes and the solvent ratio toapproximately 3.3:1 DCM/EtOH. These conditions increased the yield ofthe L-malic salt to 79.4%.

A 9:1 DCM/EtOH solvent system decreased the volume to 25 volumes. TheL-malic acid (1.05 equiv) was dissolved in 1 volume of EtOH, bringingthe total amount of solvent to 26 volumes and the final solvent ratio toapproximately 6:1 DCM/EtOH. These conditions increased the yield of theL-malic salt to 86.2%. The malic acid salt was provided in 86% yieldwhen these conditions were scaled to 8 grams. A second 8 gram reactionwas set up under these same conditions, but the slurry was cooled to 0°C. for 1.5 h before filtration. A 87.5% yield was obtained in thisreaction.

D. Stability Screening of Freebase and Selected Salts

The stabilities of the freebase of Formula I, and the HCl salt, theL-malic acid salt, and the D-malic acid salt thereof were examinedthrough weekly HPLC analysis over 4 weeks. Samples were stored at 40° C.and 75% RH. The results of the stability study are shown in Table 25.

TABLE 25 Stability of the freebase, HCl salt, L-malic acid salt, andD-malic acid salt of the compound of Formula I T = T = T = T = Absolute% Sample T = 0 1 week 2 weeks 3 weeks 4 weeks change HCl Salt 99.3%99.3% 99.3% 99.3% 99.3% 0.0% Freebase I 97.3% 97.3% 97.1% 97.1% 96.9%0.4% L-Malic 97.8% 97.7% 97.4% 97.4% 97.3% 0.5% acid salt D-Malic 97.7%97.6% 97.4% 97.2% 97.1% 0.6% acid salt

Example 4: Polymorph Salt Screens for the Compound of Formula II

A. Instrumentation and Methods of Analysis

The instruments and methods of analysis used in the polymorph screensdescribed in Examples 2 and 3 below are as follows.

X-Ray Powder Diffraction (XRPD)

XRPD analysis was carried out on a Panalytical X'pert pro, scanning thesamples between 3 and 35° 2θ. The material was gently ground and loadedonto a multi-well plate with Kapton or mylar polymer film to support thesample. The multi-well plate was then loaded into a Panalyticaldiffractometer running in transmission mode and analyzed, using thefollowing experimental conditions.

-   -   Raw Data Origin: XRD measurement (*.XRDML)    -   Scan Axis: Gonio    -   Start Position [°2θ]: 3.0066    -   End Position [°2θ]: 34.9866    -   Step Size [°2θ]: 0.0130    -   Scan Step Time [s]: 18.8700    -   Scan Type: Continuous    -   PSD Mode: Scanning    -   PSD Length [°2θ]: 3.35    -   Offset [°2θ]: 0.0000    -   Divergence Slit Type: Fixed    -   Divergence Slit Size [° ]: 1.0000    -   Measurement Temperature [° C.]: 25.00    -   Anode Material: Cu    -   K-Alpha1 [Å]: 1.54060    -   K-Alpha2 [Å]: 1.54443    -   K-Beta [Å]: 1.39225    -   K-A2/K-A1 Ratio: 0.50000    -   Generator Settings: 40 mA, 40 kV    -   Goniometer Radius [mm]: 240.00    -   Dist. Focus-Diverg. Slit [mm]: 91.00    -   Incident Beam Monochromator: No    -   Spinning: No

Polarized Light Microscopy (PLM)

The presence of crystallinity (birefringence) was determined using anOlympus BX50 polarizing microscope, equipped with a Motic camera andimage capture software (Motic Images Plus 2.0). All images were recordedusing the 20× objective, unless otherwise stated.

Thermogravimetric Analysis (TGA)

Approximately 5 mg of material was weighed into an open aluminum pan andloaded into a simultaneous thermogravimetric/differential thermalanalyzer (TG/DTA) and held at room temperature. The sample was thenheated at a rate of 10° C./min from 20° C. to 300° C. during which timethe change in sample weight was recorded along with any differentialthermal events (DTA). Nitrogen was used as the purge gas, at a flow rateof 300 cm³/min.

Differential Scanning Calorimetry (DSC)

Approximately 5 mg of material was weighed into an aluminum DSC pan andsealed nonhermetically with a pierced aluminum lid. The sample pan wasthen loaded into a Seiko DSC6200 (equipped with a cooler), cooled, andheld at 20° C. Once a stable heat-flow response was obtained, the sampleand reference were heated at scan rate of 10° C./min and the resultingheat flow response monitored.

Nuclear Magnetic Resonance (NMR)

NMR experiments were performed on a Bruker AVIIIHD spectrometer equippedwith a DCH cryoprobe operating at 500.12 MHz for protons. Experimentswere performed in deuterated solvents and each sample was prepared toapproximately 10 mM concentration.

Dynamic Vapor Sorption (DVS)

Approximately 10 mg of sample was placed into a mesh vapor sorptionbalance pan and loaded into a DVS-1 dynamic vapor sorption balance bySurface Measurement Systems. The sample was subjected to a rampingprofile from 40-90% relative humidity (RH) at 10% increments,maintaining the sample at each step until a stable weight had beenachieved (99.5% step completion). After completion of the sorptioncycle, the sample was dried using the same procedure to 0% RH and then asecond sorption cycle back to 40% RH. The weight change during thesorption/desorption cycles were plotted, allowing for the hygroscopicnature of the sample to be determined. XRPD analysis was then carriedout on any solid retained.

Gravimetric Vapor Sorption (GVS)

Approximately 10-20 mg of sample was placed into a mesh vapor sorptionbalance pan and loaded into an IGASorp Moisture Sorption Analyzerbalance by Hiden Analytical. The sample was subjected to a rampingprofile from 40-90% RH at 10% increments, maintaining the sample at eachstep until a stable weight had been achieved (98% step completion).After completion of the sorption cycle, the sample was dried using thesame procedure to 0% RH, and finally taken back to the starting point of40% RH. The weight change during the sorption/desorption cycles wereplotted, allowing for the hygroscopic nature of the sample to bedetermined.

High Performance Liquid Chromatography-Ultraviolet Detection (HPLC-UV)

-   -   Instrument: HPLC with UV detector.    -   Column: ACE Excel 3 Super C18 75 mm×4.6 mm column    -   Column Temperature: 40° C.    -   Autosampler Temperature: 25° C.    -   UV wavelength: 235 nm    -   Injection Volume: 5.0 QL    -   Flow Rate: 1.0 mL/min    -   Mobile Phase A: 0.1% TFA in water    -   Mobile Phase B: 0.1% TFA in acetonitrile    -   Gradient program:

Time (minutes) Solvent B (%) 0.00 10 15.00 90 15.10 10 20.00 10

Charged Aerosol Detection (CAD)

-   -   Instrument: HPLC with charged aerosol detection    -   Column: Thermo Acclaim P2 50×2.1 mm 3.0 QL    -   Temperature: 30° C.    -   Autosampler Temperature: Ambient    -   Injection Volume: 10 QL    -   Flow Rate: 0.5 mL/min    -   Mobile Phase A: Deionized water    -   Mobile Phase B: 100 mM, pH3.65 ammonium formate buffer    -   Gradient program:

Time (minutes) Solvent B (%) 0.00 35 2 35 12 65 15.3 65 18.6 40 19.3 3523 35

B. Initial Characterization

The compound of Formula II, prepared as described in Example 2, above,was analyzed by XRPD, TG/DTA, DSC, DVS, PLM, ¹H NMR, and HPLC andidentified as Form 1. The material appeared highly crystalline by XRPD(FIG. 62A). PLM analysis showed agglomerates of small, needle-likecrystals. A small endotherm with an onset of around 189° C. was observedby TG/DTA (FIG. 62C) followed by a sharp endotherm from an onset ofaround 199° C., relating to a melting transition. This event wasfollowed by thermal degradation. The initial endotherm was potentially asolid-solid transition followed by melting of the more stable solidForm. A small endotherm was observed in the DSC data from an onset ofaround 184° C. followed by a sharp endotherm observed from an onset ofaround 199° C. (FIG. 62B). The material appeared moderately hygroscopicby DVS (FIGS. 62D and 62E) with a weight increase of around 3.6% at 90%RH. No change in Form was observed post-DVS. The material was 99.2% pureby HPLC. ¹H NMR in d₆-DMSO showed trace DCM (0.04 eq.) and MeOH (0.06eq.) were present in the material (FIG. 62F).

C. Primary Salt Screen

A primary salt screen was conducted on the compound of Formula II usingthe acid counterions and solvent systems shown in Tables 26 and 27,below.

TABLE 26 Acids screened Hydrochloric acid Sulfuric Acid1,2-Ethanedisulfonic Methanesulfonic acid Naphthalene-2-sulfonic acid2-Hydroxyethane- acid Benzenesulfonic acid sulfonic acid L-Aspartic acidMaleic acid Ethanesulfonic acid L-Glutamic acid L-Tartaric acid CitricAcid D-Glucuronic acid L-Malic Acid D-Gluconic acid L-Lactic AcidL-Ascorbic acid Succinic Acid Oxalic acid Phosphoric acidp-Toluenesulfonic acid Fumaric Acid Hippuric acid Benzoic Acid

TABLE 27 Solvent systems Solvent system THF/water (1%) 1,4 Dioxane/water(10%) MeCN/water (20%) Acetone/water (10%) IPA/water (10%) EtOH/water(10%)

The salt screen was performed as follows. A stock aqueous solution ofacid was added to approximately 30 mg of the compound of Formula II ineach of the solvent systems listed in Table 27 above. In cases wherestock solutions could not be attained, the acid was added neat to thesolution of the compound of Formula II. Acid weights and volumes used inthe preparation of the stock solutions are shown in Table 28 below.

TABLE 28 Primary salt screen counterion stock solutions Acid StockSolution Known Acid Stock Volume of Suitable Addition Amount of SolventStock Amount Per Acid Acid (mL) Solvent Vial/Well Other Hydrochloricacid 83.5 μL 1 Water 57.1 μL Sulfuric acid 56.1 μL 1 Water 57.1 μL1,2-Ethanedisulfonic acid 234.2 mg 1 EtOH 57.1 μL Add 1 eqv. HClp-Toluenesulfonic acid 190.2 mg 1 Water 57.1 μL Methanesulfonic acid64.9 μL 1 Water 57.1 μL Naphthalene-2-sulfonic 255.8 mg 1 EtOH 57.1 μLAdd 1 eqv. HCl acid Benzenesulfonic acid 168.3 mg 1 Water 57.1 μL Oxalicacid 90.0 mg 1 Water 57.1 μL 2-Hydroxyethanesulfonic 148.1 mg 1 Water57.1 μL Add 1 eqv. HCl acid L-Aspartic acid N/A N/A N/A 7.7 mg Addedneat Maleic acid 116.1 mg 1 Water 57.1 μL Phosphoric acid 98.0 mg 1Water 57.1 μL Ethanesulfonic acid 81.6 1 Water 57.1 μL L-Glutamic acidN/A N/A N/A 8.5 mg Added neat L-Tartaric acid 150.1 mg 1 Water 57.1 μLFumaric acid N/A N/A N/A 6.5 mg Added neat Citric acid 192.1 mg 1 Water57.1 μL D- Glucuronic acid 194.1 mg 1 Water 57.1 μL L-Malic acid 134.1mg 1 Water 57.1 μL Hippuric acid N/A N/A N/A 10.3 mg Added neatD-Gluconic acid 319.0 μL 1 Water 57.1 μL L-Lactic acid 76.0 μL 1 Water57.1 μL L-Ascorbic acid 176.1 mg 1 Water 57.1 μL Benzoic acid 122.1 mg 1IPA 57.1 μL Succinic acid 118.1 mg 1 MeOH 57.1 μL

The samples were then temperature cycled between ambient (RT) and 40° C.in 4-hour cycles over 24 hours. Each mixture was filtered and isolatedsolids were analyzed (wet) by XRPD to determine crystallinity and form.Samples in which solid was not observed were stored uncapped to allowsolvent evaporation. The remaining samples were placed in an oven at 40°C. and 75% RH overnight. The samples were then reanalyzed by XRPD todetermine any changes in form or crystallinity. The observations andresults, including the different diffractogram patterns observed duringthe screen, are shown in Tables 29-53, below, and FIGS. 24A-24B through47A-47B.

TABLE 29 Hydrochloric acid observations and XRPD results SolventDioxane/ MeCN/ Acetone/ IPA/ EtOH/ THF/ water water water water waterwater (1%) (10%) (20%) (10%) (10%) (10%) Pre-cycling Slurry SlurrySlurry Slurry Slurry Slurry Post- White solid Pale orange White solidWhite solid White solid Pale yellow cycling solid solid XRPD post- Form2 Form 2 Form 2 Form 2 Form 2 Form 2 cycling XRPD post- Form 2 Form 2Form 2 Form 2 Form 2 Form 2 stability

The post-cycling and post-stability XRPD scans for hydrochloric acid areshown in FIGS. 24A and 24B, respectively.

TABLE 30 Sulfuric acid observations and XRPD results Solvent Dioxane/MeCN/ Acetone/ IPA/ EtOH/ THF/ water water water water water water (1%)(10%) (20%) (10%) (10%) (10%) Pre-cycling Slurry Slurry Slurry SlurrySlurry Slurry Post- Yellow gum Pale yellow Colorless Pale yellow Whitesolid Pale yellow cycling solution solution solution in yellow solutionsolution XRPD post- No solid No solid No solid No solid Form 3 No solidcycling XRPD post- No solid No solid No solid No solid Form 5 No solidstability

The post-cycling and post-stability XRPD scans for sulfuric acid areshown in FIGS. 25A and 25B, respectively.

TABLE 31 1,2-Ethane disulfonic acid observations and XRPD resultsSolvent Dioxane/ MeCN/ Acetone/ IPA/ EtOH/ THF/ water water water waterwater water (1%) (10%) (20%) (10%) (10%) (10%) Pre-cycling Slurry SlurrySlurry Slurry Slurry Slurry Post- Pale yellow Pale yellow Colorless Paleyellow Colorless Pale yellow cycling solution solution solution solutionsolution solution XRPD post- No solid No solid No solid No solid Nosolid No solid cycling XRPD post- No solid No solid No solid No solid Nosolid No solid stability

TABLE 32 p-Toluene sulfonic acid observations and XRPD results SolventDioxane/ MeCN/ Acetone/ IPA/ EtOH/ THF/ water water water water waterwater (1%) (10%) (20%) (10%) (10%) (10%) Pre-cycling Slurry SlurrySlurry Slurry Slurry Slurry Post- Yellow Yellow White solid White solidWhite solid White solid cycling solution solution in yellow in yellow inyellow solution solution solution XRPD post- Weak No solid No solid Form4 Amorphous Amorphous cycling diffraction XRPD post- Form 4 No solid Nosolid Form 4 Form 4 Amorphous stability

The post-cycling and post-stability XRPD scans for p-toluene sulfonicacid are shown in FIGS. 26A and 26B, respectively.

TABLE 33 Methane sulfonic acid observations and XRPD results SolventDioxane/ MeCN/ Acetone/ IPA/ EtOH/ THF/ water water water water waterwater (1%) (10%) (20%) (10%) (10%) (10%) Pre-cycling Slurry SlurrySlurry Slurry Slurry Slurry Post- Yellow Yellow Yellow White solid Whitesolid White solid cycling solution solution solution XRPD No solid Nosolid No solid Amorphous Form 2 Amorphous post- cycling XRPD No solid Nosolid No solid Amorphous Form 2 Amorphous post- stability

The post-cycling and post-stability XRPD scans for methane sulfonic acidare shown in FIGS. 27A and 27B, respectively.

TABLE 34 Naphthalene-2-sulfonic acid observations and XRPD resultsSolvent Dioxane/ MeCN/ Acetone/ IPA/ EtOH/ THF/ water water water waterwater water (1%) (10%) (20%) (10%) (10%) (10%) Pre-cycling Slurry SlurrySlurry Slurry Slurry Slurry Post- Yellow Yellow Pale yellow White solidWhite solid White solid cycling solution solution solution in yellow inyellow solution solution XRPD post- Form 5 Form 2/ Form 2/ Form 2/ Form2/ Form 2 cycling Form 5 Form 5 Form 5 Form 5 XRPD post- Form 2/ Form 2/Form 2/ Form 2/ Form 2/ Form 2 stability Form 5 Form 5 Form 5 Form 5Form 5

The post-cycling and post-stability XRPD scans fornapththalene-2-sulfonic acid are shown in FIGS. 28A and 28B,respectively.

TABLE 35 Benzene sulfonic acid observations and XRPD results SolventDioxane/ MeCN/ Acetone/ IPA/ EtOH/ THF/ water water water water waterwater (1%) (10%) (20%) (10%) (10%) (10%) Pre-cycling Slurry SlurrySlurry Slurry Slurry Slurry Post- Pale yellow White solid Pale yellowWhite solid White solid White solid cycling solution solution XRPD post-No solid No solid No solid Weak Form 2 Form 2 cycling diffraction XRPDpost- No solid No solid No solid Weak Form 2 Form 2 stabilitydiffraction

The post-cycling and post-stability XRPD scans for benzene sulfonic acidare shown in FIGS. 29A and 29B, respectively.

TABLE 36 Oxalic acid observations and XRPD results Solvent Dioxane/MeCN/ Acetone/ IPA/ EtOH/ THF/ water water water water water water (1%)(10%) (20%) (10%) (10%) (10%) Pre-cycling Slurry Slurry Slurry SlurrySlurry Slurry Post- Pale yellow White solid White solid White solidWhite solid White solid cycling solid in yellow solution XRPD post- Nosolid Form 6 No solid Amorphous Amorphous Amorphous cycling XRPD post-No solid Form 6 No solid Form 2/ Form 6 Form 6 stability Form 6

The post-cycling and post-stability XRPD scans for oxalic acid are shownin FIGS. 30A and 30B, respectively.

TABLE 37 2-Hydroxy ethanesulfonic acid observations and XRPD resultsSolvent Dioxane/ MeCN/ Acetone/ IPA/ EtOH/ THF/ water water water waterwater water (1%) (10%) (20%) (10%) (10%) (10%) Pre-cycling Slurry SlurrySlurry Slurry Slurry Slurry Post- White solid White solid White solidWhite solid White solid White solid cycling XRPD post- Form 1 Form 7Form 1 Form 1 Form 2/ Form 7 cycling (freebase)/ (freebase) (freebase)Form 7 Form 2 XRPD post- Form 1 Form 7 Form 1 Form 1 Form 2/ Form 7stability (freebase)/ (freebase)/ (freebase)/ Form 7 Form 2 Form 8 Form8

The post-cycling and post-stability XRPD scans for 2-hydroxyethanesulfonic acid are shown in FIGS. 31A and 31B, respectively.

TABLE 38 L-Aspartic acid observations and XRPD results Solvent Dioxane/MeCN/ Acetone/ IPA/ EtOH/ THF/ water water water water water water (1%)(10%) (20%) (10%) (10%) (10%) Pre-cycling Slurry Slurry Slurry SlurrySlurry Slurry Post- Yellow Pale yellow Colorless White solid White solidWhite solid cycling solid solution solution XRPD post- Form 8 Form 1Form 1 Form 1 Form 8 Form 1 cycling (freebase)/ (freebase)/ (freebase)/(freebase)/ Form 8 Form 8 Form 8 Form 8 XRPD post- Form 8 Form 1 Form 8Form 14 Form 8 Form 1 stability (freebase)/ (freebase)/ Form 8 Form 8

The post-cycling and post-stability XRPD scans for L-aspartic acid areshown in FIGS. 32A and 32B, respectively.

TABLE 39 Maleic acid observations and XRPD results Solvent Dioxane/MeCN/ Acetone/ IPA/ EtOH/ THF/ water water water water water water (1%)(10%) (20%) (10%) (10%) (10%) Pre-cycling Slurry Slurry Slurry SlurrySlurry Slurry Post- Yellow Colorless Colorless White solid White solidWhite solid cycling solid solution solution XRPD post- Weak No solid Nosolid Amorphous Amorphous Amorphous cycling diffraction XRPD post- Form15 No solid No solid Amorphous Amorphous Amorphous stability

The post-cycling and post-stability XRPD scans for maleic acid are shownin FIGS. 33A and 33B, respectively.

TABLE 40 Phosphoric acid observations and XRPD results Solvent Dioxane/MeCN/ Acetone/ IPA/ EtOH/ THF/ water water water water water water (1%)(10%) (20%) (10%) (10%) (10%) Pre-cycling Slurry Slurry Slurry SlurrySlurry Slurry Post- Yellow gum Yellow Pale yellow Pale yellow Whitesolid Yellow gum cycling solution gum gum XRPD post- Amorphous No solidNo solid Form 9 Form 10 Weak cycling diffraction XRPD post- Form 10 Nosolid No solid Form 9/ Form 10 Form 10 stability Form 10

The post-cycling and post-stability XRPD scans for phosphoric acid areshown in FIGS. 34A and 34B, respectively.

TABLE 41 Ethane sulfonic acid observations and XRPD results SolventDioxane/ MeCN/ Acetone/ IPA/ EtOH/ THF/ water water water water waterwater (1%) (10%) (20%) (10%) (10%) (10%) Pre-cycling Slurry SlurrySlurry Slurry Slurry Slurry Post- Yellow Yellow gum Pale yellow Paleyellow White solid Yellow cycling solution gum gum solution XRPD post-No solid No solid No solid No solid Amorphous No solid cycling XRPDpost- No solid No solid No solid No solid Amorphous No solid stability

The post-cycling and post-stability XRPD scans for ethane sulfonic acidare shown in FIGS. 35A and 35B, respectively.

TABLE 42 L-Glutamic acid observations and XRPD results Solvent Dioxane/MeCN/ Acetone/ IPA/ EtOH/ THF/ water water water water water water (1%)(10%) (20%) (10%) (10%) (10%) Pre-cycling Slurry Slurry Slurry SlurrySlurry Slurry Post- White solid White solid White solid White solidWhite solid White solid cycling in yellow solution XRPD post- Form 8Form 7 Form 1 Form 1 Form 1 Form 1 cycling (freebase) (freebase)(freebase)/ (freebase)/ Form 2 Form 2 XRPD post- Form 8 Form 7 Form 1Form 1 Form 1 Form 1 stability (freebase) (freebase) (freebase)/(freebase) Form 2

The post-cycling and post-stability XRPD scans for L-glutamic acid areshown in FIGS. 36A and 36B, respectively.

TABLE 43 L-Tartaric acid observations and XRPD results Solvent Dioxane/MeCN/ Acetone/ IPA/ EtOH/ THF/ water water water water water water (1%)(10%) (20%) (10%) (10%) (10%) Pre-cycling Slurry Slurry Slurry SlurrySlurry Slurry Post- White solid White solid White solid White solidWhite solid White solid cycling in yellow solution XRPD post- Weak WeakNo solid Weak Form 11 Weak cycling diffraction diffraction diffractiondiffraction XRPD post- Weak Weak No solid Weak Form 11 Weak stabilitydiffraction diffraction diffraction diffraction

The post-cycling and post-stability XRPD scans for L-tartaric acid areshown in FIGS. 37A and 37B, respectively.

TABLE 44 Fumaric acid observations and XRPD results Solvent Dioxane/MeCN/ Acetone/ IPA/ EtOH/ THF/ water water water water water water (1%)(10%) (20%) (10%) (10%) (10%) Pre-cycling Slurry Slurry Slurry SlurrySlurry Slurry Post- Pale yellow White solid Colorless White solid Whitesolid White solid cycling solid solution XRPD post- Form 12 No solid Nosolid No solid Form 8 Form 8 cycling XRPD post- Form 12 No solid Nosolid No solid Form 8 Form 8 stability

The post-cycling and post-stability XRPD scans for fumaric acid areshown in FIGS. 38A and 38B, respectively.

TABLE 45 Citric acid observations and XRPD results Solvent Dioxane/MeCN/ Acetone/ IPA/ EtOH/ THF/ water water water water water water (1%)(10%) (20%) (10%) (10%) (10%) Pre-cycling Slurry Slurry Slurry SlurrySlurry Slurry Post- Yellow Pale yellow Colorless Pale yellow White solidWhite solid cycling solid solution solution gum XRPD post- Weak No solidNo solid Weak Form 1 No solid cycling diffraction diffraction (freebase)XRPD post- Weak No solid No solid Weak Form 1 No solid stabilitydiffraction diffraction (freebase)

The post-cycling and post-stability XRPD scans for citric acid are shownin FIGS. 39A and 39B, respectively.

TABLE 46 D-Glucuronic acid observations and XRPD results SolventDioxane/ MeCN/ Acetone/ IPA/ EtOH/ THF/ water water water water waterwater (1%) (10%) (20%) (10%) (10%) (10%) Pre-cycling Slurry SlurrySlurry Slurry Slurry Slurry Post- Pale yellow Pale yellow Pale yellowWhite solid White solid Colorless cycling solution solution solutionsolution XRPD post- No solid No solid Form 1 No solid Form 8 Form 8cycling (freebase) XRPD post- No solid No solid Form 1 No solid Form 8Form 1 stability (freebase) (freebase)

The post-cycling and post-stability XRPD scans for D-glucuronic acid areshown in FIGS. 40A and 40B, respectively.

TABLE 47 L-Malic acid observations and XRPD results Solvent Dioxane/MeCN/ Acetone/ IPA/ EtOH/ THF/ water water water water water water (1%)(10%) (20%) (10%) (10%) (10%) Pre-cycling Slurry Slurry Slurry SlurrySlurry Slurry Post- Yellow Pale yellow White solid White solid Whitesolid White solid cycling solid solution XRPD post- Amorphous No solidNo solid Form 1 Form 8 Form 8 cycling (freebase) XRPD post- Amorphous Nosolid No solid Form 1 Form 8 Form 1 stability (freebase) (freebase)

The post-cycling and post-stability XRPD scans for L-malic acid areshown in FIGS. 41A and 41B, respectively.

TABLE 48 Hippuric acid observations and XRPD results Solvent Dioxane/MeCN/ Acetone/ IPA/ EtOH/ THF/ water water water water water water (1%)(10%) (20%) (10%) (10%) (10%) Pre-cycling Slurry Slurry Slurry SlurrySlurry Slurry Post- White solid White solid White solid White solidWhite solid White solid cycling in yellow solution XRPD post- Form 8Form 1 Form 1 Form 1 Form 8 Form 8 cycling (freebase)/ (freebase)(freebase) Form 8 XRPD post- Form 8 Form 1 Form 1 Form 1 Form 1 Form 1stability (freebase)/ (freebase) (freebase) (freebase)/ (freebase)/ Form8 Form 8 Form 8

The post-cycling and post-stability XRPD scans for hippuric acid areshown in FIGS. 42A and 42B, respectively.

TABLE 49 D-Gluconic acid observations and XRPD results Solvent Dioxane/MeCN/ Acetone/ IPA/ EtOH/ THF/ water water water water water water (1%)(10%) (20%) (10%) (10%) (10%) Pre-cycling Slurry Slurry Slurry SlurrySlurry Slurry Post- Yellow White solid White solid White solid Whitesolid White solid cycling solid XRPD post- Form 13 Form 7 Form 1 Form 1Form 8 Form 1 cycling (freebase) (freebase) (freebase)/ Form 8 XRPDpost- Form 13 Form 7 Form 1 Form 1 Form 1 Form 1 stability (freebase)(freebase) (freebase)/ (freebase)/ Form 8 Form 8

The post-cycling and post-stability XRPD scans for D-gluconic acid areshown in FIGS. 43A and 43B, respectively.

TABLE 50 L-Lactic acid observations and XRPD results Solvent Dioxane/MeCN/ Acetone/ IPA/ EtOH/ THF/ water water water water water water (1%)(10%) (20%) (10%) (10%) (10%) Pre-cycling Slurry Slurry Slurry SlurrySlurry Slurry Post- Pale yellow White solid White solid White solidWhite solid White solid cycling solid XRPD post- Form 13 Form 1 Form 1Form 1 Form 8 Form 8 cycling (freebase) (freebase) (freebase) XRPD post-Form 13 Form 1 Form 1 Form 1 Form 8 Form 1 stability (freebase)(freebase) (freebase) (freebase)/ Form 8

The post-cycling and post-stability XRPD scans for L-lactic acid areshown in FIGS. 44A and 44B, respectively.

TABLE 51 L-Ascorbic acid observations and XRPD results Solvent Dioxane/MeCN/ Acetone/ IPA/ EtOH/ THF/ water water water water water water (1%)(10%) (20%) (10%) (10%) (10%) Pre-cycling Slurry Slurry Slurry SlurrySlurry Slurry Post- Yellow White solid Orange Orange Yellow Yellowcycling solution in yellow solid solid solid solid solution XRPD post-No solid Form 1 Form 1 Form 1 Form 8 Form 1 cycling (freebase)(freebase) (freebase) (freebase)/ Form 8 XRPD post- No solid Form 1 Form1 Form 1 Form 1 Form 1 stability (freebase)/ (freebase) (freebase)/(freebase)/ (freebase)/ Form 2 Form 2 Form 8 Form 8

The post-cycling and post-stability XRPD scans for L-ascorbic acid areshown in FIGS. 45A and 45B, respectively.

TABLE 52 Benzoic acid observations and XRPD results Solvent Dioxane/MeCN/ Acetone/ IPA/ EtOH/ THF/ water water water water water water (1%)(10%) (20%) (10%) (10%) (10%) Pre-cycling Slurry Slurry Slurry SlurrySlurry Slurry Post- White solid White solid White solid White solidWhite solid White solid cycling XRPD post- Form 8 Form 1 Form 1 Form 1Form 8 Form 8 cycling (freebase)/ (freebase)/ (freebase) Form 8 Form 8XRPD post- Form 1 Form 1 Form 1 Form 1 Form 1 Form 1 stability(freebase)/ (freebase)/ (freebase)/ (freebase) (freebase)/ (freebase)/Form 8 Form 8 Form 8 Form 8 Form 8

The post-cycling and post-stability XRPD scans for benzoic acid areshown in FIGS. 46A and 46B, respectively.

TABLE 53 Succinic acid observations and XRPD results Solvent Dioxane/MeCN/ Acetone/ IPA/ EtOH/ THF/ water water water water water water (1%)(10%) (20%) (10%) (10%) (10%) Pre-cycling Slurry Slurry Slurry SlurrySlurry Slurry Post- White solid White solid White solid White solidWhite solid White solid cycling in yellow in yellow solution solutionXRPD post- Form 1 Form 1 Form 8 Form 1 Form 8 Weak cycling (freebase)/(freebase)/ (freebase) diffraction Form 8 Form 8 XRPD post- Form 1 Form1 Form 8 Form 1 Form 1 Form 1 stability (freebase)/ (freebase)(freebase) (freebase) (freebase) Form 8

The post-cycling and post-stability XRPD scans for succinic acid areshown in FIGS. 47A and 47B, respectively.

From the XRPD results of the primary salt screen, initial salt hits wereidentified and are shown below in Table 54.

TABLE 54 Initial salt hits from XRPD 1 Sulfate prepared in IPA/water(10%) 2 Tosylate prepared in acetone/water (10%) 3Naphthalene-2-sulfonate prepared in THF/water (10%) 4 Oxalate preparedin 1,4-dioxane/water (10%) 5 Oxalate prepared from evaporation fromTHF/water 6 Phosphate prepared in acetone/water (10%) 7 Phosphateprepared in IPA/water (10%) 8 Tartrate prepared in IPA/water (10%) 9Fumarate prepared in THF/water

Each salt was thermally analyzed using TG/DTA to identify possible saltforms. Eight of the nine initial hits were determined to be solvates orhydrates. The following results were obtained.

-   -   Sulfate (IPA/water (10%)): weight loss was observed from the        onset of heating. No distinct thermal transitions were observed.        Thermal degradation was observed around 257° C. The TG/DTA scan        of the sulfate salt is shown in FIG. 48.    -   Tosylate (acetone/water (10%)): a weight loss of approximately        6% was observed from the onset of heating followed by a second        weight loss of 3% due to solvent loss. A broad endotherm was        observed from an onset of about 28° C. followed by a broad        exotherm from an onset of around 158° C. Thermal degradation was        observed from around 192° C. The TG/DTA scan of the tosylate        salt is shown in FIG. 49.    -   Naphthalene-2-sulfonate (THF/water (10%)): weight loss was        observed from the onset of heating. No distinct thermal        transitions were observed. The TG/DTA scan of the        naphthalene-2-sulfonate salt is shown in FIG. 50.    -   Oxalate (1,4-dioxane/water (10%)): a weight loss of        approximately 0.6% was observed from the onset of heating. A        broad endotherm was observed from an onset of 194° C. followed        by thermal degradation. The TG/DTA scan of the oxalate salt is        shown in FIG. 51.    -   Oxalate (evaporation from THF/water): a broad endotherm was        observed from an onset of around 31° C. with a corresponding        weight loss of approximately 6.2%. A broad endotherm was        observed from an onset of around 100° C. with a corresponding        weight loss of approximately 1.6%. Thermal degradation was        observed from around 150° C. DSC analysis showed a broad        endotherm from an onset of around 27° C. The TG/DTA scan of the        oxalate salt is shown in FIG. 52.    -   Phosphate (acetone/water (10%)): A weight loss of approximately        3% was observed from the onset of heating. The TG/DTA scan of        the phosphate salt is shown in FIG. 53.    -   Phosphate (IPA/water (10%)): A weight loss of approximately 2.5%        was observed from the onset of heating. A small endotherm was        observed from an onset of around 162° C. Thermal degradation was        observed after 200° C. The TG/DTA scan of the phosphate salt is        shown in FIG. 54.    -   Tartrate (IPA/water (10%)): A weight loss of approximately 3.7%        was observed from the onset of heating. A small endotherm was        observed from an onset of around 173° C. followed by thermal        degradation. The TG/DTA scan of the tartrate salt is shown in        FIG. 55.    -   Fumarate (THF/water): A weight loss of 5.2% was observed from an        onset of 128° C. corresponding to a small endotherm at the same        temperature. A second small endotherm was observed from an onset        of around 190° C. with a corresponding weight loss of 2.2%. A        sharp melting endotherm was observed from an onset of 202° C.        relating to the melt of Form 2 (free base). The TG/DTA scan of        the fumarate salt is shown in FIG. 56.

D. Secondary Salt Screen

The phosphate salt prepared in IPA/water (10%) during the primary saltscreen was scaled-up and further characterization. The scale-upprocedure was as follows. 300 mg of the compound of Formula II wassuspended in 3 mL of IPA/water (10%) (10 mL/g), then 1.0 equivalent of a1M stock solution of phosphoric acid was added. The mixture was shakenand heat cycled for 4 hour increments at 40° C. overnight. The resultingsolid was filtered and washed with IPA.

The scaled-up phosphate salt material appeared crystalline by XRPD andPLM. The XRPD peaks of the phosphate salt are shown in Table 55, below,and in FIG. 23A. The diffraction pattern was consistent with dataobtained during the primary screen. Thermal analysis identified amelting point of around 167° C. (FIG. 23C). There was a weight loss ofapproximately 1.3% observed from the onset of heating. A second weightloss of approximately 1.2% was observed from an onset of around 167° C.The material appeared highly hygroscopic by GVS (FIGS. 23D and 23E),with a weight increase of approximately 14%, but no change in form orcrystallinity was observed post-GVS analysis. A broad water peak wasobserved in a ¹H NMR spectrum, indicative of salt formation (FIG. 23F).The salt was determined to be 97.7% pure by HPLC. CAD analysisdetermined the salt to have a ratio of 1.4:1, PO₄:free base.

TABLE 55 XRPD peaks of the phosphate salt Pos. FWHM Left Area d-spacingHeight Rel. Int. [°2θ] [°2θ] [cts*°2θ] [Å] [cts] [%] 3.61 0.10 151.7524.51 1503.25 100.00 6.20 0.15 87.25 14.26 576.19 38.33 7.19 0.15 57.9012.30 382.36 25.44 8.91 0.15 35.23 9.93 232.63 15.48 9.54 0.10 40.799.27 404.11 26.88 10.31 0.15 69.31 8.58 457.72 30.45 12.50 0.10 41.747.08 413.47 27.51 13.00 0.15 39.81 6.81 262.90 17.49 14.61 0.10 44.886.06 444.57 29.57 15.12 0.10 35.26 5.86 349.27 23.23 15.89 0.15 133.605.58 882.27 58.69 16.72 0.10 101.45 5.30 1004.92 66.85 17.84 0.10 92.254.97 913.84 60.79 18.22 0.10 108.09 4.87 1070.70 71.23 19.13 0.10 48.494.64 480.33 31.95 20.28 0.13 108.57 4.38 860.39 57.24 20.92 0.10 56.424.25 558.88 37.18 22.15 0.18 82.62 4.01 467.65 31.11 23.05 0.10 39.453.86 390.79 26.00 23.88 0.10 41.78 3.73 413.88 27.53 24.21 0.13 39.713.68 314.69 20.93 24.61 0.15 30.06 3.62 198.52 13.21 25.10 0.13 37.823.55 299.73 19.94 25.52 0.13 75.71 3.49 599.97 39.91 27.04 0.10 26.963.30 267.03 17.76 27.30 0.26 78.12 3.27 309.55 20.59 28.52 0.18 61.693.13 349.18 23.23 29.21 0.15 16.56 3.06 109.36 7.28 30.07 0.20 21.192.97 104.97 6.98 31.22 0.15 18.15 2.87 119.87 7.97 34.03 0.20 8.71 2.6343.12 2.87

Portions of the phosphate salt form were weighed out into 3 glass vialsfor salt stability studies. Each vial was stored for 7 days ateither: 1) ambient temperature and light; 40° C. and 75% RG; or 3) 80°C. After 7 days, each sample was analyzed by XRPD to determinecrystallinity and form. The material appeared stable under stressedconditions with no change in form or crystallinity observed after the7-day stability tests at any of the conditions tested. The materialstored at RT was found to be 98.03% pure. The material stored at 40°C./75% RH was found to be 97.44% pure. The material stored at 80° C. wasfound to be 64.96% pure. One major impurity was identified, most likelya degradant.

The thermodynamic solubility of the phosphate salt was investigated atpH 1, pH 4.5, and pH 6.8. The procedure for investigating thermodynamicsolubility was as follows. Approximately 10 mg of the phosphate salt wasweighed into a 2 mL glass vial. 2 mL of the desired buffer solution wasadded to the vial, and the resultant slurry was agitated at RTovernight. Each solution was centrifuged and observed solids werecollected. There was an insufficient amount of solids collected toanalyze the phosphate salts from the pH 1 and pH 4.5 buffers. Thephosphate salt from the pH 6.8 buffer was analyzed by XRPD and was shownto be Form 1. The mother liquor from each experiment was analyzed byHPLC to determine the solubility of each salt at each pH value. Theresults are shown in Table 56, below.

TABLE 56 Thermodynamic solubility Concentration Salt pH of buffer(mg/mL) XRPD results Phosphate 1 4.1 Insufficient solid 4.5 0.97Insufficient solid 6.8 <0.02 Form 1

Example 5: Polymorph Screens for the Compound of Formula II

A. Instrumentation and Methods of Analysis

The instruments and methods of analysis used in the polymorph screensdescribed in Examples 5 are described in Example 4, above.

B. Solvent Solubility Screen

A solvent solubility screen was conducted in 32 solvent systems asfollows.

Approximately 10 mg of the compound of Formula II was placed in each of32 vials and 5 volume aliquots of the appropriate solvent system wasadded to the appropriate vial. Between each addition, the mixture waschecked for dissolution. If no dissolution was apparent, the mixture washeated to around 40° C. and checked again. This procedure was continueduntil dissolution was observed or until 100 volumes of solvent had beenadded. Undissolved material from the solubility screen was isolated andanalyzed by XRPD. The solvent systems and the approximate solubility ofthe compound of Formula II in each solvent system tested is shown belowin Table 57, where (P) indicates partial solubility at 100 volumes.

TABLE 57 Solubility screen solvent list Approximate solubility Solvent(mg/mL) 1 1,4-Dioxane <5 (P) 2 1-Butanol <5 3 1-Propanol <5 4 Acetone <55 Acetone:Water (50%) <5 6 Anisole <5 (P) 7 CHCl₃ <5 (P) 8 Cyclohexane<5 9 Cyclohexanone <5 (P) 10 DCM <5 (P) 11 t-Butylmethyl ether N/A 12DMSO 41 13 Ethanol <5 14 EtOAc <5 15 Heptane N/A 16 IPA <5 17 Isopropylacetate N/A 18 Isopropylether N/A 19 MeCN N/A 20 MeCN:Water (20%)  5 21MEK <5 22 MeOAc <5 23 MeOH N/A 24 2-Ethoxyethanol <5 (P) 25 2-Methyl THF<5 (P) 26 MIBK <5 27 Nitromethane <5 (P) 28 NMP 17 29 THF <5 (P) 30THF:Water (1%) 48 31 Toluene N/A 32 Water <5

The material was found to have low solubility in the majority of solventsystems investigated with moderate solubility observed in only 3 solventsystems (DMSO, NMP, and THF/water (1%)).

Observed solids were filtered and analyzed by XRPD to determine form andcrystallinity. The results are shown in Table 58, below. FIG. 57 showsthe XRPD scans of the observed solids.

TABLE 58 XRPD results for observed solids Solvent Form 1 1,4-DioxaneInsufficient solid 2 1-Butanol Form 7 3 1-Propanol Form 8 4 AcetoneAmorphous 5 Acetone:Water (50%) Amorphous 6 Anisole Insufficient solid 7CHCl₃ Form 1/Form 13 8 Cyclohexane Form 1 9 Cyclohexanone Insufficientsolid 10 DCM Insufficient solid 11 t-Butylmethyl ether Form 1 12 DMSOAmorphous 13 Ethanol Form 8 14 EtOAc Weak diffraction 15 Heptane Form 116 IPA Form 2 17 Isopropyl acetate Form 1 18 Isopropylether Form 1 19MeCN Form 1 20 MeCN:Water (20%) Form 2/Form 13 21 MEK Insufficient solid22 MeOAc Form 7 23 MeOH Form 1 24 2-Ethoxyethanol Insufficient solid 252-Methyl THF Form 7 26 MIBK Form 1 27 Nitromethane Form 1 28 NMPInsufficient solid 29 THF Form 13 30 THF:Water (1%) Form 8 31 TolueneForm 1 32 Water Form 2/Form 13

C. Primary Polymorph Screen

A primary polymorph screen was conducted using the solvent systems shownin Table 59, below, selected using the data from the solvent solubilityscreen described above.

TABLE 59 Primary polymorph screen solvent list Solvent 1 Acetone 2Acetone/water (50%) 3 Anisole 4 1-Butanol 5 2-Butanone 6 Chloroform 7Cyclohexane 8 Cyclohexanone 9 Dichloromethane 10 1,4-Dioxane 11 Ethanol12 Ethyl acetate 13 Dimethyl sulfoxide 14 MeCN/water (20%) 15 Methylacetate 16 2-Ethoxyethanol 17 Nitromethane 18 Methylisobutyl ketone 192-Methyl THF 20 2-Propanol 21 1-Propanol 22 Tetrahydrofuran 23 N-Methylpyrrolidone 24 THF/water (1%) 25 Water

1. Temperature Cycling (Maturation) Experiments

Slurries for temperature cycling (maturation) experiments were preparedusing the solvent systems from Table 59, above, as follows.Approximately 40 mg of crystalline compound of Formula II was suspendedin the appropriate solvent system. In any cases where dissolutionoccurred, additional material was added until a slurry was obtained. Theresultant slurries were subjected to successive 4-hour heat-cool cyclesbetween 40° C. and RT for 72 hours.

After temperature cycling, each mixture was filtered and isolated solidswere analyzed (wet) by XRPD to determine crystallinity and form. Thesamples were then stored at 40° C. and 75% RH overnight and reanalyzedby XRPD to determine any changes in form or crystallinity. Results areshown in Table 60, below, and FIGS. 58A-58D.

TABLE 60 Temperature cycling (maturation) experiments Post-temperatureSolvent Observations cycling Post-stability tests 1,4-Dioxane Solid Weakdiffraction Form 1 1-Butanol Solid Weak diffraction Form 1 1-PropanolSolid Form 8 Form 1 Acetone Solid Weak diffraction Form 1 Acetone:WaterSolid Form 1 Form 1 (50%) Anisole Solid Form 1 Form 1 CHCl₃ SolidAmorphous Form 1 Cyclohexane Solid Form 1 Form 1 Cyclohexanone SolidWeak diffraction Form 1 DCM Solid Amorphous Form 1 DMSO Solid Form 1Form 1 Ethanol Solid Weak diffraction Form 1 EtOAc Solid Form 1 Form 1IPA Solid Form 8 Form 1 MeCN:Water Solid Weak diffraction Form 1 (20%)MEK Solid Weak diffraction Form 1 MeOAc Solid Weak diffraction Form 12-Ethoxyethanol Solid Weak diffraction Form 1 2-Methyl THF Solid Weakdiffraction Form 1 MIBK Solid Form 1 Form 1 Nitromethane Solid Weakdiffraction Form 1 NMP Insufficient Insufficient solid Insufficientsolid solid THF Solid Weak diffraction Form 1 THF:Water (1%) Solid Weakdiffraction Form 1/Form 8 Water Solid Form 1 Form 1

The supernatant from each experiment was split equally into 3 vials forevaporation, crash cooling, and anti-solvent addition experiments(described below).

2. Evaporation Experiments

For evaporation experiments, sub-samples of supernatant from theslurries containing the compound of Formula II were transferred tovials. The vials were then uncapped and allowed to evaporate at ambienttemperature. Any material recovered was analyzed by XRPD, with no priordrying. Results are shown in Table 61, below, and FIG. 59.

TABLE 61 Evaporation experiments Solvent Solid Observations Form1,4-Dioxane Yes Pale yellow solid Form 17 1-Butanol No N/A N/A1-Propanol No N/A N/A Acetone Yes White solid Form 1/Form 8Acetone:Water Yes White needle-like crystals N/A (50%) Anisole Yes Whitesolid N/A CHCl₃ Yes Yellow solid Form 1/Form 8 Cyclohexane No N/A N/ACyclohexanone No N/A N/A DCM Yes White solid Form 1 DMSO Yes White solidForm 1 Ethanol Yes White solid N/A EtOAc Yes White solid Amorphous IPANo N/A N/A MeCN:Water (20%) Yes White needle-like crystals Form 1 MEKYes White solid Amorphous MeOAc Yes White solid Form 1 2-EthoxyethanolYes White solid Form 1 2-Methyl THF Yes White solid N/A MIBK No N/A N/ANitromethane Yes Pale yellow solid Form 1 NMP No N/A N/A THF Yes Yellowsolid Form 1/Form 8 THF:Water (1%) No N/A N/A Water No N/A N/A

3. Crash Cooling Experiments

Crash cooling experiments were performed at 5° C. and −20° C.Sub-samples of supernatant from the slurries containing the compound ofFormula II were transferred into vials and left to cool at 5° C. in therefrigerator. When enough material was obtained (by precipitation), thematerial was analyzed, with no prior drying, by XRPD. For samples inwhich precipitation had not occurred, the samples were transferred to afreezer and left to cool at −20° C. When enough material was obtained,the material was analyzed, with no prior drying, by XRPD. The results ofthe crash cooling experiments conducted at 5° C. are shown in Table 62,below, and FIG. 60. No solids were observed in the samples stored at−20° C. after 10 days.

TABLE 62 Crash cooling experiments (5° C.) Solvent Solid TemperatureObservations Form 1,4-Dioxane Yes 2° C. White solid Amorphous 1-ButanolNo N/A N/A N/A 1-Propanol No N/A N/A N/A Acetone Yes 2° C. White solidForm 1/Form 8 Acetone:Water No N/A (50%) Anisole Yes 2° C. White solidForm 18 CHCl₃ No N/A N/A N/A Cyclohexane No N/A N/A N/A CyclohexanoneYes 2° C. White solid Amorphous DCM Yes 2° C. White solid N/A DMSO N/AN/A N/A N/A Ethanol No N/A N/A N/A EtOAc Yes 2° C. White solid AmorphousIPA No N/A N/A N/A MeCN:Water Yes 2° C. White needle- Form 1 (20%) likecrystals MEK Yes 2° C. White solid Amorphous MeOAc Yes 2° C. White solidAmorphous 2-Ethoxyethanol Yes 2° C. White solid Amorphous 2-Methyl THFNo N/A N/A N/A MIBK No N/A N/A N/A Nitromethane Yes 2° C. White needle-Amorphous like crystals NMP No N/A N/A N/A THF No N/A N/A N/A THF:Water(1%) No N/A N/A N/A Water No N/A N/A N/A

4. Anti-Solvent Addition Experiments

Anti-solvent addition experiments were performed by transferringsub-samples of supernatant from the slurries containing the compound ofFormula II into vials and adding anti-solvent stepwise to the saturatedsolutions. When enough material was obtained, the material was analyzed,with no prior drying, by XRPD. The solvents used in the anti-solventaddition experiments are shown in Table 63, below. The results of theanti-solvent addition experiments are shown in Table 64, below, and FIG.61.

TABLE 63 Anti-solvent addition experiments solvents and anti-solventsSample Solvent Anti-solvent 1 Acetone MTBE 2 Acetone/water (50%) MTBE 3Anisole MTBE 4 1-Butanol MTBE 5 2-Butanone MTBE 6 Chloroform MTBE 7Cyclohexane MTBE 8 Cyclohexanone Heptane 9 Dichloromethane MTBE 101,4-Dioxane DIPE 11 Ethanol DIPE 12 Ethyl acetate MTBE 13 Dimethylsulfoxide N/A 14 MeCN/water (20%) MTBE 15 Methyl acetate MTBE 162-Ethoxyethanol Water 17 Nitromethane MTBE 18 Methylisobutyl ketone DIPE19 2-Methyl THF Water 20 2-Propanol MTBE 21 1-Propanol MTBE 22Tetrahydrofuran MTBE 23 N-Methyl pyrrolidone Water 24 THF/water (1%)MTBE 25 Water MTBE

TABLE 64 Anti-solvent addition experiments Anti- Vol. of anti- Solventsolvent solvent Solid Observations Form 1,4-Dioxane DIPE 0.5 Yes Whitesolid Form 1 1-Butanol MTBE 1 Yes White solid N/A 1-Propanol MTBE 1.5 NoN/A N/A Acetone MTBE 1 Yes White solid Form 1/Form 8 Acetone:Water MTBE1 Yes White needle-like N/A (50%) crystals Anisole Water 1 Yes Whitesolid N/A CHCl₃ MTBE 1.5 Yes White solid Form 1/Form 8 Cyclohexane MTBE1 No N/A N/A Cyclohexanone Heptane 1 Yes White solid Amorphous DCM MTBE1 Yes White solid N/A DMSO N/A N/A No N/A N/A Ethanol DIPE 1 No N/A N/AEtOAc MTBE 1 No N/A N/A IPA MTBE 1 No N/A N/A MeCN:Water (20%) MTBE 1 NoN/A N/A MEK MTBE 1 Yes White solid N/A MeOAc MTBE 1 Yes White solidAmorphous 2-Ethoxyethanol Water 1 Yes White solid Amorphous 2-Methyl THFWater 1 No N/A N/A MIBK DIPE 1 No N/A N/A Nitromethane MTBE 1 No N/A N/ANMP Water 1 Yes Yellow needle-like Form 1 crystals THF MTBE 1 Yes Whitesolid Amorphous THF:Water (1%) MTBE 1 No N/A N/A Water MTBE 1 No N/A N/A

D. Secondary Polymorph Screen

Four forms were identified during the primary polymorph solvent screen:Form 1, Form 2, Form 7, and Form 8. Each form was prepared on a 300 mgscale and was fully characterized by) (RFD, TG/DTA, DSC, GVS withpost-GVS)(RFD, ¹H NMR spectroscopy, HPLC-UV, and PLM.

Seven-day stability tests were also conducted as follows. A portion ofeach polymorph form was weighed out into 3 glass vials. Each vial wasstored for 7 days at either: 1) ambient temperature and light; 40° C.and 75% RH; or 3) 80° C. After 7 days, each sample was analyzed by XRPDto determine crystallinity and form. These additional studies indicatedthat Form 1 was the most stable form of the compound of Formula IIidentified during the polymorph screen. Forms 2 and 7 were determined tobe hydrated forms of the compound of Formula II that, upon heating,dehydrate to Form 1. Form 8 was identified as an IPA solvate which, uponheating, desolvates to Form 1. No change in appearance was observedafter 7 days for any of the samples and no change in form was observedby XRPD after 7 days for any of the samples, though the sample of Form 8that was stored at 40° C./75% RH appeared to have a higher amorphouscontent.

1. Form 1

Form 1 was the most stable form of the compound of Formula II identifiedduring the polymorph screen. Form 1 was prepared on a 300 mg scale asfollows. Approximately 500 mg of the compound of Formula II free basewas weighed into a 20 mL scintillation vial. Ethyl acetate (4 mL) wasadded to the vial and the resultant slurry was temperature cycledbetween 40° C. and room temperature for 72 hours. The sample wasfiltered and the collected material was dried overnight under vacuum at40° C.

Post-drying XRPD (40° C. under vacuum) of the material appeared to beForm 1. The XRPD peaks of Form 1 are shown in Table 65, below, and FIG.19A. The material was crystalline. Highly birefringent agglomerates ofparticles were observed by PLM. A small endothermic event was observedby DSC (FIG. 19B) from an onset of around 190° C. A melting transitionwas observed from an onset of around 203° C. A small endothermic eventwas observed by TG/DTA (FIG. 19C) from an onset of around 190° C. with acorresponding weight loss of 0.4%. A melting transition was observedfrom an onset of around 204° C. with a corresponding weight loss of0.2%. Degradation was observed after the melting transition. Thematerial appeared moderately hygroscopic by GVS (FIGS. 19D and 19E) witha weight increase of 2.6% at 90% RH. No change in form was observed byXRPD post-GVS analysis. Trace amounts of solvent were observed by ¹H NMR(FIG. 19F). The spectra obtained was consistent with that of thereceived material (compound of Formula II).

TABLE 65 XRPD peaks of Form 1 Pos. FWHM Left Area d-spacing Height Rel.Int. [°2θ] [°2θ] [cts*°2θ] [Å] [cts] [%] 4.95 0.19 24.33 17.84 97.486.05 8.23 0.19 61.56 10.74 246.62 15.32 9.75 0.19 82.45 9.07 330.3420.52 12.77 0.19 41.33 6.93 165.59 10.28 13.80 0.19 35.96 6.41 144.068.95 14.77 0.19 46.81 5.99 187.52 11.65 15.51 0.19 79.80 5.71 319.7119.86 16.53 0.12 267.93 5.36 1610.16 100.00 17.11 0.19 44.95 5.18 180.0811.18 17.82 0.12 93.39 4.97 561.24 34.86 18.86 0.16 220.48 4.70 1060.0265.83 22.34 0.19 29.93 3.98 119.89 7.45 22.81 0.19 29.57 3.89 118.477.36 23.75 0.19 145.46 3.74 582.78 36.19 24.99 0.12 52.20 3.56 313.7119.48 25.32 0.12 99.61 3.52 598.63 37.18 25.59 0.19 132.87 3.48 532.3533.06 25.96 0.19 158.21 3.43 633.84 39.36 26.56 0.12 46.49 3.35 279.3617.35 27.07 0.37 96.42 3.29 193.14 12.00 28.28 0.12 55.67 3.15 334.5720.78 28.86 0.19 64.48 3.09 258.35 16.04 32.32 0.31 26.00 2.77 62.493.88 33.33 0.37 32.74 2.69 65.59 4.07

2. Form 2

Form 2 was isolated from Form 7 and was considered to be a hydrated formof the compound of Formula II that dehydrated to Form 1 upon heating.Form 2 was prepared on a 300 mg scale as follows. Approximately 500 mgof the compound of Formula II free base was weighed into a 20 mLscintillation vial. Ethanol/water (4 mL, 10%) was added to the vial andthe resultant slurry was temperature cycled between 40° C. and roomtemperature for 72 hours. The sample was filtered and the collectedmaterial was dried on the filter bed overnight.

XRPD analysis of the slurry material (scaled-up) was inconsistent withthe small scale material observed in the primary salt screen—thediffractogram was consistent with the Form 7 pattern. XRPD analysisconducted post-drying (on filter bed) on the material appeared to beForm 1. The XRPD peaks of Form 2 are shown in Table 66, below, and FIG.20A. The material was crystalline. Highly birefringent agglomerates ofparticles were observed by PLM. A small endothermic event was observedby DSC (FIG. 20B) from an onset of around 192° C. A melting transitionof Form 1 was observed from an onset of around 204° C. A weight loss of0.7% was observed by TG/DTA (FIG. 20C) from the onset of heating. Asmall endothermic event was observed from an onset of around 194° C.with a corresponding weight loss of 0.2%. The solid-solid transition andmelting transition of Form 1 was observed from an onset of around 205°C. Degradation was observed after the melting transition. The materialappeared moderately hygroscopic by GVS (FIGS. 20D and 20E) with a weightincrease of 2% at 90% RH. No change in form was observed by XRPDpost-GVS analysis. Trace amounts of solvent were observed by ¹H NMR (notshown). The spectra obtained was consistent with that of the receivedmaterial (compound of Formula II).

TABLE 66 XRPD peaks of Form 2 Pos. FWHM Left Area d-spacing Height Rel.Int. [°2θ] [°2θ] [cts*°2θ] [Å] [cts] [%] 6.19 0.15 92.45 14.28 610.559.20 8.91 0.10 34.21 9.93 338.85 5.11 10.29 0.15 65.01 8.60 429.31 6.4713.43 0.41 18.88 6.59 46.76 0.70 13.87 0.15 11.28 6.38 74.48 1.12 14.680.15 76.40 6.03 504.57 7.60 15.12 0.10 194.68 5.86 1928.53 29.06 15.870.15 16.00 5.58 105.68 1.59 16.50 0.15 76.75 5.37 506.86 7.64 16.79 0.1039.53 5.28 391.55 5.90 17.14 0.10 28.80 5.17 285.27 4.30 17.42 0.1343.66 5.09 346.03 5.21 17.82 0.10 669.86 4.98 6635.61 100.00 18.14 0.1064.77 4.89 641.58 9.67 18.72 0.15 11.62 4.74 76.73 1.16 19.09 0.15 11.114.65 73.40 1.11 20.38 0.10 80.18 4.36 794.26 11.97 21.08 0.15 100.834.21 665.90 10.04 22.10 0.15 85.85 4.02 566.98 8.54 22.81 0.10 44.293.90 438.70 6.61 23.37 0.10 62.75 3.81 621.59 9.37 24.20 0.10 182.813.68 1810.90 27.29 24.61 0.10 61.83 3.62 612.45 9.23 25.00 0.13 45.503.56 360.61 5.43 25.48 0.10 27.06 3.50 268.10 4.04 26.14 0.10 10.42 3.41103.22 1.56 27.21 0.10 49.21 3.28 487.46 7.35 27.40 0.10 29.76 3.26294.85 4.44 27.97 0.13 22.74 3.19 180.20 2.72 29.03 0.15 32.60 3.08215.30 3.24 29.36 0.13 49.11 3.04 389.19 5.87 29.63 0.13 56.71 3.01449.38 6.77 29.98 0.13 44.37 2.98 351.62 5.30 30.50 0.10 13.45 2.93133.23 2.01 31.20 0.10 58.94 2.87 583.89 8.80 31.66 0.10 19.31 2.83191.32 2.88 32.22 0.13 43.31 2.78 343.20 5.17 32.61 0.20 9.60 2.75 47.530.72 34.09 0.13 20.42 2.63 161.81 2.44 34.46 0.10 25.96 2.60 257.14 3.88

3. Form 7

Form 7 was considered to be a hydrated form of the compound of FormulaII that dehydrated to Form 1 upon heating. Form 7 was prepared on a 300mg scale as follows. Approximately 500 mg of the compound of Formula IIfree base was weighed into a 20 mL scintillation vial. 4 mL of1,4-dioxane/water (10%) was added to the vial and the resultant slurrywas temperature cycled between 40° C. and room temperature for 72 hours.The sample was filtered and the collected material was dried on thefilter bed overnight.

XRPD analysis of the slurry material was consistent with the small scalematerial identified in the primary salt screen. XRPD post-drying (onfilter bed) of the material appeared to be Form 1. The XRPD peaks ofForm 7 are shown in Table 67, below, and FIG. 21A. The material wascrystalline. Highly birefringent agglomerates of particles were observedby PLM. An endotherm was observed by DSC (FIG. 21B) from an onset ofaround 147° C. A small endotherm was observed from an onset of around196° C. relating to the transition observed in Form 1. A meltingtransition of Form 1 was observed from an onset of around 205° C. Anendotherm was observed by TG/DTA (FIG. 21C) from an onset of around 147°C. with a corresponding weight loss of approximately 7% relating to theloss of solvent. The weight loss equated to approximately 2 equivalentsof water present. A small endotherm was observed from an onset of around196° C. relating to the transition observed in Form 1. The meltingtransition of Form 1 was observed from an onset of around 206° C. Thematerial showed slight hygroscopicity by GVS (FIGS. 21D and 21E) with aweight increase of 0.8% at 90% RH, and no change in form was observed byXRPD post-GVS analysis. Trace amounts of solvent were observed by 41 NMR(FIG. 21F).

TABLE 67 XRPD peaks of Form 7 Pos. FWHM Left Area d-spacing Height Rel.Int. [°2θ] [°2θ] [cts*°2θ] [Å] [cts] [%] 4.88 0.15 124.64 18.10 823.1014.40 6.19 0.15 19.85 14.28 131.10 2.29 8.59 0.15 142.39 10.30 940.3516.45 9.87 0.15 176.65 8.96 1166.58 20.41 10.31 0.15 28.54 8.58 188.503.30 11.62 0.10 34.57 7.62 342.41 5.99 12.58 0.10 36.56 7.03 362.18 6.3414.14 0.15 140.39 6.27 927.13 16.22 14.84 0.15 143.38 5.97 946.87 16.5715.77 0.26 452.10 5.62 1791.38 31.34 16.58 0.15 865.48 5.35 5715.58100.00 17.26 0.10 199.94 5.14 1980.57 34.65 18.04 0.10 290.40 4.922876.67 50.33 18.97 0.10 237.60 4.68 2353.62 41.18 19.34 0.15 399.144.59 2635.93 46.12 19.91 0.10 334.71 4.46 3315.58 58.01 21.35 0.10180.69 4.16 1789.93 31.32 21.84 0.10 138.81 4.07 1375.00 24.06 23.340.10 258.86 3.81 2564.23 44.86 24.28 0.13 108.82 3.67 862.39 15.09 25.120.10 217.06 3.55 2150.16 37.62 25.73 0.10 158.86 3.46 1573.65 27.5326.04 0.10 95.40 3.42 945.00 16.53 27.11 0.10 121.21 3.29 1200.73 21.0127.51 0.15 243.26 3.24 1606.48 28.11 28.47 0.20 64.59 3.14 319.91 5.6028.84 0.13 73.98 3.10 586.26 10.26 30.18 0.15 26.71 2.96 176.40 3.0931.17 0.15 38.53 2.87 254.43 4.45 31.61 0.10 27.96 2.83 276.98 4.8532.81 0.15 32.51 2.73 214.67 3.76 33.58 0.20 21.74 2.67 107.69 1.88

4. Form 8

Form 8 was identified as an isopropyl alcohol (IPA) solvate whichdesolvates upon heating to Form 1. Form 8 was prepared on a 300 mg scaleas follows. Approximately 500 mg of the compound of Formula II free basewas weighed into a 20 mL scintillation vial. 4 mL of IPA was added tothe vial and the resultant slurry was temperature cycled between 40° C.and room temperature for 72 hours. The sample was filtered and thecollected material was dried overnight under vacuum at 40° C.

XRPD analysis of the slurry material was consistent with the small scalematerial observed in the primary polymorph and salt screens. XRPDpost-drying (40° C. under vacuum) analysis of the material appeared tobe Form 8. The XRPD peaks of Form 8 are shown in Table 68, below, andFIG. 22A. The material was crystalline. Highly birefringent agglomeratesof particles were observed by PLM. An endotherm was observed by DSC(FIG. 22B) from an onset of around 168° C. A small endotherm wasobserved from an onset of around 190° C. relating to the transitionobserved in Form 1. A melting transition of Form 1 was observed from anonset of around 203° C. An endotherm was observed by TG/DTA (FIG. 22C)from an onset of around 165° C. with a corresponding weight loss ofapproximately 4% relating to the loss of solvent. The weight lossequated to approximately 0.5 equivalents of IPA present in the sample. Asmall endotherm was observed from an onset of around 191° C. relating tothe transition observed in Form 1. A melting transition of Form 1 wasobserved from an onset of around 205° C. The material appearedmoderately hygroscopic by GVS (FIGS. 22D and 22E) with a weight increaseof 2.6% at 90% RH. A higher amorphous content was observed by XRPDpost-GVS analysis. 0.5 equivalents of IPA were observed by ¹H NMR (FIG.22F).

TABLE 68 XRPD peaks of Form 8 Pos. FWHM Left Area d-spacing Height Rel.Int. [°2θ] [°2θ] [cts*°2θ] [Å] [cts] [%] 6.19 0.15 92.45 14.28 610.559.20 8.91 0.10 34.21 9.93 338.85 5.11 10.29 0.15 65.01 8.60 429.31 6.4713.43 0.41 18.88 6.59 46.76 0.70 13.87 0.15 11.28 6.38 74.48 1.12 14.680.15 76.40 6.03 504.57 7.60 15.12 0.10 194.68 5.86 1928.53 29.06 15.870.15 16.00 5.58 105.68 1.59 16.50 0.15 76.75 5.37 506.86 7.64 16.79 0.1039.53 5.28 391.55 5.90 17.14 0.10 28.80 5.17 285.27 4.30 17.42 0.1343.66 5.09 346.03 5.21 17.82 0.10 669.86 4.98 6635.61 100.00 18.14 0.1064.77 4.89 641.58 9.67 18.72 0.15 11.62 4.74 76.73 1.16 19.09 0.15 11.114.65 73.40 1.11 20.38 0.10 80.18 4.36 794.26 11.97 21.08 0.15 100.834.21 665.90 10.04 22.10 0.15 85.85 4.02 566.98 8.54 22.81 0.10 44.293.90 438.70 6.61 23.37 0.10 62.75 3.81 621.59 9.37 24.20 0.10 182.813.68 1810.90 27.29 24.61 0.10 61.83 3.62 612.45 9.23 25.00 0.13 45.503.56 360.61 5.43 25.48 0.10 27.06 3.50 268.10 4.04 26.14 0.10 10.42 3.41103.22 1.56 27.21 0.10 49.21 3.28 487.46 7.35 27.40 0.10 29.76 3.26294.85 4.44 27.97 0.13 22.74 3.19 180.20 2.72 29.03 0.15 32.60 3.08215.30 3.24 29.36 0.13 49.11 3.04 389.19 5.87 29.63 0.13 56.71 3.01449.38 6.77 29.98 0.13 44.37 2.98 351.62 5.30 30.50 0.10 13.45 2.93133.23 2.01 31.20 0.10 58.94 2.87 583.89 8.80 31.66 0.10 19.31 2.83191.32 2.88 32.22 0.13 43.31 2.78 343.20 5.17 32.61 0.20 9.60 2.75 47.530.72 34.09 0.13 20.42 2.63 161.81 2.44 34.46 0.10 25.96 2.60 257.14 3.88

Example 6: Polymorph of the Compound of Formula III

The compound of Formula III, prepared as described in Example 2 wasanalyzed by XRPD, DSC, and ¹H NMR and identified as Form A. The X-raypowder diffraction scan of Form A is shown in FIG. 63A. The X-ray powderdiffraction peaks of Form A is shown in Table 69. An endothermic eventwas observed by DSC from an onset of around 83° C. (FIG. 63B).

TABLE 69 XRPD peaks of Form A of the Compound of Formula III Theta-2Intensity Theta-2 Intensity 2.02 4930 13.42 3244 2.74 2840 14 1121 3.122219 14.5 1495 4.24 2468 15.24 3953 4.66 5782 15.86 2721 5.1 1247 16.681776 6.24 1061 17.3 9498 6.76 3997 17.72 1523 7.16 905 18.76 5838 7.74827 19.2 9152 8.7 811 20.24 3605 9.22 1144 20.96 1217 9.34 1419 21.922319 9.72 815 22.1 1894 10.2 776 23.2 1306 10.56 755 23.86 5921 11.5 74024.1 2517 11.86 715 25.24 1657 12.66 1246 25.48 2717 13.24 1713 25.71941

Example 7: Polymorph of the Compound of Formula IV

A. Initial Characterization

The compound of Formula IV, prepared as described in Example 2 wasanalyzed by XRPD, DSC, and ¹H NMR and identified as Form A. The X-raypowder diffraction scan of Form A is shown in FIG. 64A. The X-ray powderdiffraction peaks of Form A is shown in Table 70. An endothermic eventwas observed by DSC from an onset of around 149° C. (FIG. 64B).

TABLE 70 XRPD peaks of Form A of the Compound of Formula IV 2-Theta d(Å) Height H % 8.32 10.62 373.20 100.00 9.05 9.77 8.90 2.40 10.11 8.740.90 0.20 10.65 8.30 1.20 0.30 11.49 7.70 30.90 8.30 12.25 7.22 27.507.40 13.03 6.79 24.40 6.50 13.51 6.55 3.30 0.90 14.08 6.29 0.70 0.2014.38 6.16 1.40 0.40 14.68 6.03 4.30 1.20 15.60 5.68 3.00 0.80 16.275.44 265.60 71.20 16.64 5.32 94.40 25.30 16.89 5.25 5.00 1.40 17.57 5.0424.40 6.50 18.08 4.90 42.00 11.30 18.61 4.76 39.00 10.50 18.85 4.7041.00 11.00 19.15 4.63 23.80 6.40 19.35 4.58 92.10 24.70 19.80 4.48 3.601.00 20.04 4.43 49.20 13.20 20.28 4.37 30.50 8.20 20.45 4.34 59.60 16.0020.90 4.25 0.30 0.10 21.59 4.11 68.30 18.30 21.92 4.05 120.80 32.4022.69 3.92 13.90 3.70 23.01 3.86 4.10 1.10 23.34 3.81 6.80 1.80 23.813.73 11.40 3.10 24.41 3.64 26.50 7.10 25.19 3.53 11.20 3.00 25.71 3.467.30 2.00 26.06 3.42 17.40 4.70 27.04 3.29 12.40 3.30 28.21 3.16 3.400.90 28.78 3.10 1.10 0.30 29.56 3.02 1.20 0.30 30.41 2.94 17.90 4.8031.04 2.88 3.00 0.80 31.87 2.81 8.40 2.20 32.10 2.79 21.40 5.70 33.122.70 1.30 0.40 33.61 2.66 3.60 1.00 34.16 2.62 5.80 1.50 34.81 2.58 4.101.10 35.11 2.55 6.20 1.60 36.76 2.44 2.40 0.60 37.00 2.43 2.40 0.6037.42 2.40 1.80 0.50 37.84 2.38 2.10 0.60 39.36 2.29 2.00 0.50

B. Solubility Screening

The solubility of the compound of Formula IV (freebase) was tested indifferent solvent mixtures as shown in Table 71.

TABLE 71 Solubility of the compound of Formula IV (freebase) atapproximately 25° C. Solvent mg/mL Acetone 42.9 Acetone:water (1:1) 45.8Acetone:heptane (1:1) 6.1 Butanol 16.6 MTBE 5.5 Ethanol 5.2 EtOH:water(1:1) 51 EtOH:water (90:10) 55 Ethyl acetate 21.0 Heptane 0.0 MEK 30.7IPA 7.0 ACN 21.2 Chloroform >59 Chloroform:heptane (1:1) 11.7 DCM >70p-dioxane >62 Hexane 0.0 Methanol >74 THF >37 THF:heptane (30:70) 0.6Water 0.007

C. Polymorph Screening

Further polymorph screen was conducted with expanding crystallizationtechniques such as cooling and solvent/anti-solvent precipitation. Theresults are shown in Table 72.

TABLE 72 Polymorph screening of the compound of Formula IV XRPD SolventCrystallization Technique Result Acetone Slurry at ~25° C. for ~3 daysForm A Acetone/water Vapor diffusion at RT Form A Acetone:water (1:1)Slurry at ~25° C. for ~3 days Form A Acetone/heptane Vapor diffusion atRT Form A Acetone:heptane (2:1) Dissolved API at ~55° C., cooled Form Aslowly to RT without stirring Acetone:heptane (1:1) Slurry at ~25° C.for ~3 days Form A CAN Slurry at ~25° C. for ~3 days Form A 2-butanolSlurry at ~25° C. for ~3 days Form A 2-butanol Slurry at ~5° C. for ~3days Form A Chloroform Slow evaporation at RT — Chloroform/heptane Vapordiffusion at RT Form A Chloroform:heptane (1:1) Slurry at ~25° C. for ~3days Form A DCM Slow evaporation at RT — p-dioxane Fast evaporation atRT — EtOH Slurry at ~25° C. for ~3 days Form A EtOH:water (1:1) Slurryat ~25° C. for ~3 days Form A EtOH:water (90:10) Slurry at ~25° C. for~3 days Form B EtOAc Slurry at ~25° C. for ~3 days Form A EtOAc Slurryat ~5° C. for ~3 days Form A EtOAc Dissolved API at ~70° C., cooled FormA slowly to RT Heptane Slurry at ~25° C. for ~3 days Form A HexaneSlurry at ~25° C. for ~3 days Form A IPA Slurry at ~25° C. for ~3 daysForm A IPA Dissolved API at ~70° C., cooled Form A slowly to RT withoutstirring MeOH Slow evaporation at RT — MeOH/water Vapor diffusion at RT,oil — MEK Slurry at ~25° C. for ~3 days, — insufficient solids MTBESlurry at ~25° C. for ~3 days Form A THF Slow evaporation at RT —THF:heptane (30:70) Slurry at ~25° C. for ~3 days Form A THF:heptane(1:4) Solvent/anti-solvent Form A precipitation, precipitation andoiling observed, let stir at RT for ~1 day THF:water (1:4)Solvent/anti-solvent — precipitation, precipitation and minor oilingobserved, stirred at ~5° C., oil persisted THF/heptane Vapor diffusionat RT Form A Water Slurry at ~25° C. for ~3 days Form A

Form B: A new XRPD pattern was observed from a slurry in ethanol/water(90:10) as shown in table 72, above. This new pattern has beendesignated Form B.

Form B was prepared on ˜65 mg scale by slurrying a compositioncomprising Form A and 1 mL of 9:1 EtOH:H₂O at 25° C. for ˜36 hours. Thesolid was isolated by centrifuge filtration (nylon filter, bench-topmini-centrifuge, 8000 rpm, five minutes) and then dried in a vacuum ovenwith N2 bleed at room temperature overnight (˜18 hours) before XRPD andDSC.

Form B was analyzed further by XRPD and DSC. The XRPD data is shown inFIG. 65A and Table 73. The DSC thermogram of Form B is shown in FIG.65B. An endothermic event was observed by DSC from an onset of around162° C., about 12° C. higher than that of Form A. An overlay of theX-ray powder diffraction scans of polymorphs A and B of the compound ofFormula IV is shown in FIG. 66.

TABLE 73 XRPD peaks of Form B of the compound of Formula IV 2-Theta d(Å) Height H % 4.85 18.19 20.60 5.30 7.45 11.86 390.20 100.00 7.69 11.4824.90 6.40 9.70 9.11 127.30 32.60 9.94 8.89 88.80 22.80 13.74 6.44142.80 36.60 14.54 6.09 94.00 24.10 14.89 5.94 1.90 0.50 15.43 5.7419.40 5.00 15.76 5.62 3.30 0.90 16.31 5.43 20.70 5.30 16.89 5.24 149.0038.20 17.15 5.17 14.30 3.70 17.39 5.09 10.60 2.70 17.79 4.98 10.80 2.8018.08 4.90 25.60 6.60 18.48 4.80 20.60 5.30 19.44 4.56 126.00 32.3019.90 4.46 127.60 32.70 20.80 4.27 2.50 0.60 21.31 4.17 100.00 25.6021.31 4.17 100.00 25.60 21.44 4.14 62.10 15.90 22.21 4.00 2.60 0.7022.41 3.96 15.80 4.00 22.80 3.90 27.70 7.10 23.05 3.86 9.80 2.50 23.533.78 9.50 2.40 24.25 3.67 23.30 6.00 24.50 3.63 27.00 6.90 24.91 3.5710.00 2.60 25.35 3.51 3.80 1.00 25.71 3.46 0.90 0.20 26.31 3.39 15.704.00 26.74 3.33 2.60 0.70 26.94 3.31 4.20 1.10 27.39 3.25 31.00 7.9028.24 3.16 13.10 3.40 28.92 3.08 9.20 2.40 29.56 3.02 0.70 0.20 30.042.97 3.70 0.90 31.12 2.87 2.10 0.50 31.76 2.82 5.70 1.50 31.99 2.80 2.100.50 32.27 2.77 1.00 0.30 32.98 2.71 2.80 0.70 33.61 2.66 10.20 2.6034.19 2.62 2.00 0.50 34.68 2.58 1.20 0.30 35.11 2.55 1.70 0.40 35.662.52 1.70 0.40 35.84 2.50 2.80 0.70 36.32 2.47 1.30 0.30 38.05 2.36 2.900.70 39.00 2.31 1.80 0.50 39.21 2.30 3.00 0.80

Example 8: RET Enzyme Assay

The compounds of Formula I-IV were screened for their ability to inhibitwildtype and V804M mutant RET kinase using CisBio's HTRF® KinEASE™-TKassay technology. Briefly, N-terminal GST tagged recombinant human RETcytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Catalog No.14-570M) or N-terminal GST tagged recombinant human V804M mutant RETcytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; CatalogNo. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, partof Catalog No. 62TKOPEC) and 1 mM ATP along with test compound in abuffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl₂, 0.01% TritonX-100, and 2% DMSO in a volume of 8 μL. The compound was typicallyprepared in a threefold serial dilution in DMSO and added to the assayto give the appropriate final concentration. After a 30-minuteincubation at 22° C., the reaction was quenched by adding 8 of quenchsolution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRFdetection buffer (all from CisBio, part of Cat. No. 62TKOPEC). After a 1hour incubation at 22° C., the extent of reaction was determined using aPerkinElmer EnVision multimode plate reader via HTRF dual wavelengthdetection, and the percent of control (POC) was calculated using aratiometric emission factor. 100 POC was determined using no testcompounds and 0 POC was determined using pre-quenched control reactions.The POC values were fit to a 4 parameter logistic curve, and the IC₅₀ isdefined as the concentration of inhibitor at which the POC equals 50 forthe fitted curve. The compound of Formula I was found to have IC₅₀values of 4.5 nM and 18.9 nM for inhibiting wildtype RET enzyme andV804M mutant RET kinase, respectively. The compound of Formula II wasfound to have IC₅₀ values of 14.0 nM and 24.1 nM for inhibiting wildtypeRET enzyme and V804M mutant RET kinase, respectively. The compound ofFormula III was found to have IC₅₀ values of 29.9 nM and 59.0 nM forinhibiting wildtype RET enzyme and V804M mutant RET kinase,respectively. The compound of Formula IV was found to have IC₅₀ valuesof 17.0 nM and 121.7 nM for inhibiting wildtype RET enzyme and V804Mmutant RET kinase, respectively.

Example 9: RET Cell Assay

The cellular potency of a compound inhibiting RET kinase was determinedin HEK-293 cells expressing a Kif5b-RET fusion protein. Briefly, HEK-293cells expressing a Kif5b-RET fusion protein were plated at 50Kcells/well in 96-well poly-D-lysine-coated plates the day prior to theassay. The cells were incubated for 1 hour with test compound in DMEM(Dulbecco's Modified Eagle Medium) at a final DMSO concentration of0.5%. The compound of Formula I-IV was typically prepared in a threefoldserial dilution in DMSO and added to the assay to give the appropriatefinal concentration. After 1 hour the media was removed, the cells werefixed with 3.8% formaldehyde for 20 min, washed with PBS, andpermeabilized for 10 min with 100% methanol. The plates were then washedwith PBS-0.05% Tween20, and blocked with LI-COR Blocking solution(LI-COR catalog #927-40000) for 1 hour. Plates were washed withPBS-0.05% Tween20, then incubated with anti-phospho-RET(Tyr1062) (SantaCruz catalog # sc-20252-R) antibody and anti-GAPDH (Millipore catalog #MAB374) antibody for 2 hours. The plates were washed with PBS-0.05%Tween20, and incubated with anti-rabbit 680 (Molecular Probes catalogNo. A21109) and anti-mouse 800 (LI-COR catalog No. 926-32210) secondaryantibodies for 1 hour. All antibodies were diluted in LI-COR Blockcontaining 0.05% Tween. The plates were washed with PBS-0.05% Tween20,100 μL PBS was added to each well, and the plates were read on a LI-CORAerius fluorescent plate reader. The phospho-RET signal was normalizedto the GAPDH signal. 100 POC (percent of control) was determined usingno test compounds and 0 POC was determined using 1 μM of a controlinhibitor. The POC values were fit to a 4 parameter logistic curve. TheIC₅₀ value is the point where the curve crosses 50 POC. The compound ofFormula I was found to have an IC₅₀ value of 4.0 nM for inhibiting RETkinase in HEK-293 cells expressing a Kif5b-RET fusion protein. Thecompound of Formula II was found to have an IC₅₀ value of 4.2 nM forinhibiting RET kinase in HEK-293 cells expressing a Kif5b-RET fusionprotein. The compound of Formula III was found to have an IC₅₀ value of10.9 nM for inhibiting RET kinase in HEK-293 cells expressing aKif5b-RET fusion protein. The compound of Formula IV was found to havean IC₅₀ value of 16.5 nM for inhibiting RET kinase in HEK-293 cellsexpressing a Kif5b-RET fusion protein.

What is claimed is:
 1. A crystalline form of a compound of Formula IIhaving the formula

wherein the crystalline form is selected from the group consisting ofForm 1, characterized by having an X-ray powder diffraction (XRPD)pattern comprising peaks at °2θ values of 16.5±0.2, 18.9±0.2, and26.0±0.2; Form 2, characterized by having an X-ray powder diffraction(XRPD) pattern comprising peaks at °2θ values of 15.1±0.2, 17.8±0.2, and24.2±0.2; Form 7, characterized by having an X-ray powder diffraction(XRPD) pattern comprising peaks at °2θ values of 16.6±0.2, 18.0±0.2, and19.9±0.2; and Form 8, characterized by having an X-ray powderdiffraction (XRPD) pattern comprising peaks at °2θ values of 15.1±0.2,17.8±0.2, and 24.2±0.2.
 2. The crystalline form of claim 1, wherein thecrystalline form is Form 1, characterized by having an X-ray powderdiffraction (XRPD) pattern comprising peaks at °2θ values of: a)16.5±0.2, 18.9±0.2, 23.8±0.2, 25.3±0.2, and 26.0±0.2; b) 16.5±0.2,17.8±0.2, 18.9±0.2, 23.8±0.2, 25.3±0.2, 25.6±0.2, 26.0±0.2, and28.3±0.2; or c) 9.8±0.2, 16.5±0.2, 17.8±0.2, 18.9±0.2, 23.8±0.2,25.0±0.2, 25.3±0.2, 25.6±0.2, 26.0±0.2, and 28.3±0.2.
 3. A method oftreating a RET-associated cancer in a subject, the method comprisingadministering to a subject identified or diagnosed as having aRET-associated cancer a therapeutically effective amount of a compoundof claim
 1. 4. The method of claim 3, wherein the RET-associated canceris selected from the group consisting of: lung cancer, papillary thyroidcancer, medullary thyroid cancer, differentiated thyroid cancer,recurrent thyroid cancer, refractory differentiated thyroid cancer,multiple endocrine neoplasia type 2A or 2B (MEN2A or MEN2B,respectively), pheochromocytoma, parathyroid hyperplasia, breast cancer,colorectal cancer, papillary renal cell carcinoma, ganglioneuromatosisof the gastroenteric mucosa, and cervical cancer.
 5. The method of claim4, wherein the cancer is RET fusion lung cancer or medullary thyroidcancer.
 6. The method of claim 4, wherein the lung cancer is small celllung carcinoma, non-small cell lung cancer, bronchioles lung cellcarcinoma, or lung adenocarcinoma.
 7. The crystalline form of claim 1,wherein the crystalline form is Form 2, characterized by having an X-raypowder diffraction (XRPD) pattern comprising peaks at °2θ values of: a)15.1±0.2, 17.8±0.2, 20.4±0.2, 21.1±0.2, and 24.2±0.2; b) 15.1±0.2,17.8±0.2, 18.1±0.2, 20.4±0.2, 21.1±0.2, 23.4±0.2, 24.2±0.2, and24.6±0.2; or c) 6.2±0.2, 15.1±0.2, 17.8±0.2, 18.1±0.2, 20.4±0.2,21.1±0.2, 23.4±0.2, 24.2±0.2, 24.6±0.2, and 31.2±0.2.
 8. The crystallineform of claim 1, wherein the crystalline form is Form 7, characterizedby having an X-ray powder diffraction (XRPD) pattern comprising peaks at°2θ values of: a) 16.6±0.2, 18.0±0.2, 19.3±0.2, 19.9±0.2, and 23.3±0.2;b) 16.6±0.2, 17.3±0.2, 18.0±0.2, 19.0±0.2, 19.3±0.2, 19.9±0.2, 23.3±0.2,and 25.1±0.2; or c) 15.8±0.2, 16.6±0.2, 17.3±0.2, 18.0±0.2, 19.0±0.2,19.3±0.2, 19.91±0.2, 21.4±0.2, 23.3±0.2, and 25.1±0.2.
 9. Thecrystalline form of claim 1, wherein the crystalline form is anisopropyl alcohol solvate Form 8, characterized by having an X-raypowder diffraction (XRPD) pattern comprising peaks at °2θ values of: a)15.1±0.2, 17.8±0.2, 20.4±0.2, 21.1±0.2, and 24.2±0.2; b) 15.1±0.2,17.8±0.2, 18.1±0.2, 20.4±0.2, 21.1±0.2, 23.4±0.2, 24.2±0.2, and24.6±0.2; or c) 6.2±0.2, 15.1±0.2, 17.8±0.2, 18.1±0.2, 20.4±0.2,21.1±0.2, 23.4±0.2, 24.2±0.2, 24.6±0.2, and 31.2±0.2.